Protease Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to protease variants and methods for obtaining protease variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel protease variants exhibitingalterations relative to the parent protease in one or more propertiesincluding: wash performance, detergent stability and/or storagestability. The variants of the invention are suitable for use in e.g.cleaning or detergent compositions, such as laundry detergentcompositions and dish wash compositions, including automatic dish washcompositions. The present invention also relates to isolated DNAsequences encoding the variants, expression vectors, host cells, andmethods for producing and using the variants of the invention.

2. Description of the Related Art

Enzymes have been used within the detergent industry as part of washingformulations for many decades. Proteases are from a commercialperspective the most relevant enzyme in such formulations, but otherenzymes including lipases, amylases, cellulases, hemicellulases ormixtures of enzymes are also often used. To improve the cost and/or theperformance of proteases there is an ongoing search for proteases withaltered properties, such as increased activity at low temperatures,increased stability, increased specific activity at a given pH, alteredCa²⁺ dependency, increased stability in the presence of other detergentingredients (e.g. bleach, surfactants etc.) etc. One family ofproteases, which are often used in detergents, are the subtilases. Thisfamily has previously been further grouped into 6 different sub-groupsby Siezen R J and Leunissen J A M, 1997, Protein Science, 6, 501-523.One of these sub-groups is the Subtilisin family which includessubtilases such as BPN′, subtilisin 309 (SAVINASE®, Novozymes A/S),subtilisin Carlsberg (ALCALASE®, Novozymes A/S), subtilisin S41 (asubtilase from the psychrophilic Antarctic Bacillus TA41, Davail S etal. 1994, The Journal of Biological Chemistry, 269(26), 99. 17448-17453)and subtilisin S39 (a subtilase from the psychrophilic AntarcticBacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11), pp.1271-1279). TY145 is a subtilase from Bacillus sp. TY145, NCIMB 40339,which was first described in WO 92/17577 (Novozymes A/S) and in thelater application WO2004/067737 (Novozymes A/S) disclosing thethree-dimensional structure and the use of protein engineering to alterfunctionality of a TY-145 subtilase.

SUMMARY OF THE INVENTION

The present invention relates to protease variants, comprising analteration at one or more (e.g., several) positions corresponding topositions 171, 173, 175 or 179 of SEQ ID NO: 3, wherein the variant haveprotease activity and wherein the variants has an amino acid sequencewhich is at least 75% identical to the mature polypeptide of SEQ ID NO:2 or to SEQ ID NO: 3.

The present invention relates to a method for obtaining a proteasevariant, comprising introducing into a parent protease a substitution atone or more positions 171, 173, 175 or 179 of SEQ ID NO: 3, wherein thevariant has an amino acid sequence which is at least 75%, at least 80%,at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3;and recovering the variant. The present invention also relates toisolated polynucleotides encoding the variants; nucleic acid constructs,vectors, and host cells comprising the polynucleotides; and methods ofproducing the variants.

Overview of Sequences Listing

SEQ ID NO: 1=is the DNA sequence of TY-145 protease isolated fromBacillus sp.

SEQ ID NO: 2=is the amino acid sequence as deduced from SEQ ID NO: 1.

SEQ ID NO: 3=is the amino acid sequence of the mature TY145.

DEFINITIONS

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996,237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999,264, 610-650; respectively. The term “subtilases” refer to a sub-groupof serine protease according to Siezen et al., Protein Engng. 4 (1991)719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serineproteases or serine peptidases is a subgroup of proteases characterisedby having a serine in the active site, which forms a covalent adductwith the substrate. Further the subtilases (and the serine proteases)are characterised by having two active site amino acid residues apartfrom the serine, namely a histidine and an aspartic acid residue. Thesubtilases may be divided into 6 sub-divisions, i.e. the Subtilisinfamily, the Thermitase family, the Proteinase K family, the Lantibioticpeptidase family, the Kexin family and the Pyrolysin family. The term“protease activity” means a proteolytic activity (EC 3.4). Proteases ofthe invention are endopeptidases (EC 3.4.21). There are several proteaseactivity types: The three main activity types are: trypsin-like wherethere is cleavage of amide substrates following Arg or Lys at P1,chymotrypsin-like where cleavage occurs following one of the hydrophobicamino acids at P1, and elastase-like with cleavage following an Ala atP1. For purposes of the present invention, protease activity isdetermined according to the procedure described in “Materials andMethods” below. The protease variants of the present invention have atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of theprotease activity of the mature polypeptide with SEQ ID NO: 3.

The term “parent” or protease parent means a protease to which analteration is made to produce the enzyme variants of the presentinvention. Thus the parent is a protease having the identical amino acidsequence of said variant but not having the alterations at one or moreof said specified positions. It will be understood, that in the presentcontext the expression “having identical amino acid sequence” relates to100% sequence identity. The parent may be a naturally occurring(wild-type) polypeptide or a variant thereof. In a particular embodimentthe parent is a protease with at least 70%, at least 72%, at least 73%,at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, e.g. at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6 or100% identity to a polypeptide with SEQ ID NO: 3.

The term “protease variant” means a protease having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, preferably substitution, at one or more (or one or several)positions compared to its parent which is a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. A substitutionmeans a replacement of an amino acid occupying a position with adifferent amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding amino acids e.g. 1to 10 amino acids, preferably 1-3 amino acids adjacent to an amino acidoccupying a position.

The term “isolated variant” means a variant that is modified by the handof man. In one aspect, the variant is at least 1% pure, e.g., at least5% pure, at least 10% pure, at least 20% pure, at least 40% pure, atleast 60% pure, at least 80% pure, and at least 90% pure, as determinedby SDS-PAGE.

The term “isolated polynucleotide” means a polynucleotide that ismodified by the hand of man. In one aspect, the isolated polynucleotideis at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least20% pure, at least 40% pure, at least 60% pure, at least 80% pure, atleast 90% pure, and at least 95% pure, as determined by agaroseelectrophoresis. The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “wild-type protease” means a protease expressed by a naturallyoccurring organism, such as a bacterium, archaea, yeast, fungus, plantor animal found in nature. An example of a wild-type protease is TY-145i.e. the mature polypeptide of SEQ ID NO: 2 i.e. amino acids 1 to 311 orthe mature polypeptide with SEQ ID NO: 3.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In one aspect, the mature polypeptide correspondsto the amino acid sequence with SEQ ID NO: 3.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having protease activity. In oneaspect, the mature polypeptide coding sequence is nucleotides 331 to1263 of SEQ ID NO: 1 based on the SignalP (Nielsen et al., 1997, ProteinEngineering 10: 1-6)] that predicts nucleotides 1 to 81 of SEQ ID NO: 1is the signal peptide.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aprokaryotic or eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of its polypeptide product. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic. The termnucleic acid construct is synonymous with the term “expression cassette”when the nucleic acid construct contains the control sequences requiredfor expression of a coding sequence of the present invention.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence.

The term “control sequences” means all components necessary for theexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native or foreign to thepolynucleotide encoding the variant or native or foreign to each other.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

The term “expression” includes any step involved in the production ofthe variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to additional nucleotides that provide for its expression.

The term “transcription promoter” is used for a promoter which is aregion of DNA that facilitates the transcription of a particular gene.Transcription promoters are typically located near the genes theyregulate, on the same strand and upstream (towards the 5′ region of thesense strand).

The term “transcription terminator” is used for a section of the geneticsequence that marks the end of gene or operon on genomic DNA fortranscription.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, and the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and either 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “improved property” means a characteristic associated with avariant that is improved compared to the parent or compared to aprotease with SEQ ID NO: 3, or compared to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions. Such improvedproperties include, but are not limited to, wash performance, proteaseactivity, thermal activity profile, thermostability, pH activityprofile, pH stability, substrate/cofactor specificity, improved surfaceproperties, substrate specificity, product specificity, increasedstability, improved stability under storage conditions, and chemicalstability.

The term “improved protease activity” is defined herein as an alteredprotease activity (as defined above) e.g. by increased proteinconversion of a protease variant displaying an alteration of theactivity relative (or compared) to the activity of the parent protease,or compared to a protease with SEQ ID NO: 3, or relative to a proteasehaving the identical amino acid sequence of said variant but not havingthe alterations at one or more of said specified positions.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process and reflects the stability of theprotease variant according to the invention as a function of time e.g.how much activity is retained when the protease variant is kept insolution in particular in a detergent solution. The stability isinfluenced by many factors e.g. pH, temperature, detergent compositione.g. amount of builder, surfactants etc. The protease stability may bemeasured using the assay described in example 2. The term “improvedstability” or “increased stability” is defined herein as a variantprotease displaying an increased stability in solutions, relative to thestability of the parent protease, relative to a protease having theidentical amino acid sequence of said variant but not having thealterations at one or more of said specified positions or relative toSEQ ID NO: 3. The terms “improved stability” and “increased stability”includes “improved chemical stability”, “detergent stability” or“improved detergent stability.

The term “improved chemical stability” is defined herein as a variantenzyme displaying retention of enzymatic activity after a period ofincubation in the presence of a chemical or chemicals, either naturallyoccurring or synthetic, which reduces the enzymatic activity of theparent enzyme. Improved chemical stability may also result in variantsbeing more able to catalyze a reaction in the presence of suchchemicals. In a particular aspect of the invention the improved chemicalstability is an improved stability in a detergent, in particular in aliquid detergent. The term “detergent stability” or “improved detergentstability is in particular an improved stability of the proteaseactivity when a protease variant of the present invention is mixed intoa liquid detergent formulation, especially into a liquid detergentformulation according to table 1 and then stored at temperatures between15 and 50° C., e.g. 20° C., 30° C. or 40° C.

The term “improved thermal activity” means a variant displaying analtered temperature-dependent activity profile at a specific temperaturerelative to the temperature-dependent activity profile of the parent orrelative to a protease with SEQ ID NO: 3. The thermal activity valueprovides a measure of the variant's efficiency in enhancing catalysis ofa hydrolysis reaction over a range of temperatures. A more thermo activevariant will lead to an increase in enhancing the rate of hydrolysis ofa substrate by an enzyme composition thereby decreasing the timerequired and/or decreasing the enzyme concentration required foractivity. Alternatively, a variant with a reduced thermal activity willenhance an enzymatic reaction at a temperature lower than thetemperature optimum of the parent defined by the temperature-dependentactivity profile of the parent.

The term “improved wash performance” is defined herein as a proteasevariant according to the invention displaying an improved washperformance relative to the wash performance of the parent protease,relative to a protease with SEQ ID NO: 3 or relative to a proteasehaving the identical amino acid sequence of said variant but not havingthe alterations at one or more of said specified positions e.g. byincreased stain removal. The term “wash performance” includes washperformance in laundry but also e.g. in dish wash. The wash performancemay be quantified as described under the definition of “washperformance” herein.

The term “detergent composition”, includes unless otherwise indicated,granular or powder-form all-purpose or heavy-duty washing agents,especially cleaning detergents; liquid, gel or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid (HDL) types;liquid fine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, cleaningbars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos,bathroom cleaners; hair shampoos and hair-rinses; shower gels, foambaths; metal cleaners; as well as cleaning auxiliaries such as bleachadditives and “stain-stick” or pre-treat types. The terms “detergentcomposition” and “detergent formulation” are used in reference tomixtures which are intended for use in a wash medium for the cleaning ofsoiled objects. In some embodiments, the term is used in reference tolaundering fabrics and/or garments (e.g., “laundry detergents”). Inalternative embodiments, the term refers to other detergents, such asthose used to clean dishes, cutlery, etc. (e.g., “dishwashingdetergents”). It is not intended that the present invention be limitedto any particular detergent formulation or composition. The term“detergent composition” is not intended to be limited to compositionsthat contain surfactants. It is intended that in addition to thevariants according to the invention, the term encompasses detergentsthat may contain, e.g., surfactants, builders, chelators or chelatingagents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

The term “fabric” encompasses any textile material. Thus, it is intendedthat the term encompass garments, as well as fabrics, yarns, fibers,non-woven materials, natural materials, synthetic materials, and anyother textile material.

The term “textile” refers to woven fabrics, as well as staple fibers andfilaments suitable for conversion to or use as yarns, woven, knit, andnon-woven fabrics. The term encompasses yarns made from natural, as wellas synthetic (e.g., manufactured) fibers. The term, “textile materials”is a general term for fibers, yarn intermediates, yarn, fabrics, andproducts made from fabrics (e.g., garments and other articles).

The term “non-fabric detergent compositions” include non-textile surfacedetergent compositions, including but not limited to compositions forhard surface cleaning, such as dishwashing detergent compositions, oraldetergent compositions, denture detergent compositions, and personalcleansing compositions.

The term “effective amount of enzyme” refers to the quantity of enzymenecessary to achieve the enzymatic activity required in the specificapplication, e.g., in a defined detergent composition. Such effectiveamounts are readily ascertained by one of ordinary skill in the art andare based on many factors, such as the particular enzyme used, thecleaning application, the specific composition of the detergentcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, and the like. The term “effective amount” of aprotease variant refers to the quantity of protease variant describedhereinbefore that achieves a desired level of enzymatic activity, e.g.,in a defined detergent composition.

The term “water hardness” or “degree of hardness” or “dH” or “° dH” asused herein refers to German degrees of hardness. One degree is definedas 10 milligrams of calcium oxide per litre of water.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,detergent concentration, type of detergent and water hardness, actuallyused in households in a detergent market segment.

The term “adjunct materials” means any liquid, solid or gaseous materialselected for the particular type of detergent composition desired andthe form of the product (e.g., liquid, granule, powder, bar, paste,spray, tablet, gel, or foam composition), which materials are alsopreferably compatible with the protease variant enzyme used in thecomposition. In some embodiments, granular compositions are in “compact”form, while in other embodiments, the liquid compositions are in a“concentrated” form.

The term “stain removing enzyme” as used herein, describes an enzymethat aids the removal of a stain or soil from a fabric or a hardsurface. Stain removing enzymes act on specific substrates, e.g.,protease on protein, amylase on starch, lipase and cutinase on lipids(fats and oils), pectinase on pectin and hemicellulases onhemicellulose. Stains are often depositions of complex mixtures ofdifferent components which either results in a local discolouration ofthe material by itself or which leaves a sticky surface on the objectwhich may attract soils dissolved in the washing liquor therebyresulting in discolouration of the stained area. When an enzyme acts onits specific substrate present in a stain the enzyme degrades orpartially degrades its substrate thereby aiding the removal of soils andstain components associated with the substrate during the washingprocess. For example, when a protease acts on a grass stain it degradesthe protein components in the grass and allows the green/brown colour tobe released during washing.

The term “reduced amount” means in this context that the amount of thecomponent is smaller than the amount which would be used in a referenceprocess under otherwise the same conditions. In a preferred embodimentthe amount is reduced by, e.g., at least 5%, such as at least 10%, atleast 15%, at least 20% or as otherwise herein described.

The term “low detergent concentration” system includes detergents whereless than about 800 ppm of detergent components is present in the washwater. Asian, e.g., Japanese detergents are typically considered lowdetergent concentration systems.

The term “medium detergent concentration” system includes detergentswherein between about 800 ppm and about 2000 ppm of detergent componentsis present in the wash water. North American detergents are generallyconsidered to be medium detergent concentration systems.

The term “high detergent concentration” system includes detergentswherein greater than about 2000 ppm of detergent components is presentin the wash water. European detergents are generally considered to behigh detergent concentration systems.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 3 is used to determine the corresponding amino acidresidue in another protease. The amino acid sequence of another proteaseis aligned with the mature polypeptide disclosed in SEQ ID NO: 3, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the mature polypeptide disclosed in SEQ ID NO:3 is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 5.0.0 or later. The parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 3 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP super families of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letters amino acid abbreviations are employed.Amino acid positions are indicated with #₁, #₂, etc.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of serine at position #₁ withtryptophan is designated as “Ser#₁Trp” or “S#₁W”. Multiple mutations areseparated by addition marks (“+”) or by commas (,), e.g.,“Ser#₁Trp+“Ser#₂Pro” or S#₁W, S#₂P, representing substitutions atpositions #₁ and #₂ of serine (S) with tryptophan (W) and proline (P),respectively. If more than one amino acid may be substituted in a givenposition these are listed in brackets, such as [X] or {X}. Thus if bothTrp and Lys according to the invention may be substituted instead of theamino acid occupying at position #₁ this is indicated as X#₁ {W, K} orX#₂ [W, K] where the X indicate that different proteases may be parente.g. such as a protease with SEQ ID NO 3 or a protease having at least70% identity hereto. Thus in some cases the variants are represented as#₁ {W, K} or X#₂P indicating that the amino acids to be substituted varydepending on the parent.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofserine at position #₁ is designated as “Ser#₁*” or “S#₁*”. Multipledeletions are separated by addition marks (“+”) or commas, e.g.,“Ser#₁*+Ser#₂*” or “S#₁*, S#₂*”.

Insertions: The insertion of an additional amino acid residue such ase.g. a lysine after G#₁ may be indicated by: Gly#₁GlyLys or G#₁GK.Alternatively insertion of an additional amino acid residue such aslysine after G#₁ may be indicated by: *#aL. When more than one aminoacid residue is inserted, such as e.g. a Lys, and Ala after #₁ this maybe indicated as: Gly#₁GlyLysAla or G#₁GKA. In such cases, the insertedamino acid residue(s) may also be numbered by the addition of lower caseletters to the position number of the amino acid residue preceding theinserted amino acid residue(s), in this example: *#₁aK *#₁bA.

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”) or by commas (,), e.g.,“Ser#₁Trp+Ser#₂Pro” or “S#₁W, S#₂P” representing a substitution ofserine at positions #₁ and #₂ with tryptophan and proline, respectivelyas described above.

Different alterations: Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Ser#₁Trp, Lys” or S#₁W, K represents a substitution of serine atposition #₁ with tryptophan or lysine. Thus, “Ser#₁Trp, Lys+Ser#₂Asp”designates the following variants: “Ser#₁Trp+Ser#₂Pro”,“Ser#₁Lys+Ser#₂Pro” or S#₁W, K+S#₂D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns protease variants, comprising asubstitution of one or more amino acids in the loop corresponding topositions 171, 173, 175 or 179 of SEQ ID NO: 3, wherein the variant hasa sequence identity to SEQ ID NO: 3 of at least 75% and less than 100%,and wherein the variant has protease activity. Previously unanticipated,the inventors have found that protease variants containing one or moresubstitutions corresponding to the positions 171, 173, 175 or 179 of SEQID NO: 3 have improved stability in detergent compared to a proteasehaving the identical amino acid sequence of said variant but not havingthe substitution(s) at one or more of said specified positions orcompared to a protease with SEQ ID NO: 3. The amino acids correspondingto positions 171, 173, 175 or 179 of SEQ ID NO: 3 form part of an activesite loop corresponding to positions 170 to 180 of SEQ ID NO: 3, whichdefines parts of the S1 substrate binding pocket, see FIG. 1. Thus thepresent invention provides protease variants, comprising a substitutionat one or more (e.g., several) positions corresponding to positions 171,173, 175 or 179, wherein the variant has protease activity. New proteasevariants containing one or more substitution(s) in the loop region170-180 (SEQ ID NO: 3 numbering), were generated and tested forstability in detergent as described in “Material and Methods” and theinventors demonstrate that one or more substitutions of one or moreamino acid at a position corresponding to positions 171, 173, 175 or 179of the mature polypeptide SEQ ID NO: 3 significantly improved thedetergent stability compared to a protease having the identical aminoacid sequence of said variant but not having a substitution at one ormore of said specified positions or compared to a protease with SEQ IDNO: 3.

Surprisingly the variants according to the invention may in addition toimproved stability also have improved wash performance. Thus in apreferred embodiment the variants according to the invention haveimproved the detergent stability and/or improved wash performancecompared to a protease having the identical amino acid sequence of saidvariant but not having a substitution at one or more of said specifiedpositions or compared to a protease with SEQ ID NO:3. In a preferredembodiment the protease variant comprises a substitution of one or moreamino acids in the loop corresponding to positions 171, 173, 175 or 179of SEQ ID NO: 3, wherein the variant has at least 70% identity to theprotease with SEQ ID NO: 3 (Bacillus sp. TY145). Thus one aspect of theinvention relates to a protease variant, comprising a substitution atone or more positions corresponding to positions 171, 173, 175 or 179 ofSEQ ID NO: 3, wherein the variant has an amino acid sequence which is atleast 75% identical to SEQ ID NO 3; and recovering the variant. Thus,the invention relates to such variant comprising substitution of one ormore amino acids in the loop corresponding to positions 171, 173, 175 or179 of SEQ ID NO: 3, wherein the variant has at least 70%, such as atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%,e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, atleast 99.5%, at least 99.6, but less than 100%, sequence identity to SEQID NO: 3. In one embodiment, the variant is a polypeptide encoded by apolynucleotide having at least 70% identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or a sequence encoding the maturepolypeptide of SEQ ID NO: 2. In one embodiment the variant according tothe invention is a polypeptide encoded by a polynucleotide having atleast 70% identity e.g., such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95% identity, at least 96%, at least 97%, atleast 98%, or at least 99% e.g. at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6, but less than100%, sequence identity to the mature polynucleotide of SEQ ID NO: 1.

A particular embodiment, concerns a protease variant, comprising asubstitution at one or more positions corresponding to positions 171,173, 175 or 179 of SEQ ID NO: 3, wherein the variant is a variant of aparent protease which has at least 70%, such as at least 71%, at least72%, at least 73%, at least 74%, such as at least 75%, e.g., such as atleast 76% at least 77% at least 78% at least 79% at least 80%, at least81% at least 82% at least 83% at least 84% at least 85%, at least 86% atleast 87% at least 88% at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% e.g. at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6, or100% sequence identity to SEQ ID NO: 3. In one particular embodiment theprotease variant is a TY-145 (SEQ ID NO 3) variant comprising asubstitution of one or more amino acids in the loop corresponding topositions 171, 173, 175 or 179 of SEQ ID NO: 3. In another embodimentthe invention relates to a variant comprising a substitution at two,three, four or five positions corresponding to positions 171, 173, 175,179 or 180 of SEQ ID NO: 3. A preferred embodiment concerns a proteasevariant, comprising substitution of one or more amino acids in the loopcorresponding to positions 171, 173, 175 or 179 of SEQ ID NO: 3, whereinthe variant has at least 70%, such as at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94% at least 95% identity, at least 96%, at least97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, sequence identity to SEQ ID NO: 3. A particularly preferredembodiment concerns a protease variant comprising a substitution of oneor more amino acid selected from the group consisting of Cys, Val, Gln,Thr, Glu, His, Lys, Met, Asn, Tyr, Ala, Pro and Trp in the loopcorresponding to positions 171, 173, 175 or 179 of SEQ ID NO: 3. Aparticular embodiment concerns a protease variant comprising asubstitution of one or more amino acid selected from the groupconsisting of Cys, Val, Gln, Thr, Glu, His, Lys, Met, Asn, Tyr, Ala, Proand Trp in the loop corresponding to positions 171, 173, 175 or 179 ofSEQ ID NO: 3, wherein the variant has at least 70% identity to SEQ IDNO: 3 such as at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94% atleast 95% identity, at least 96%, at least 97%, at least 98%, or atleast 99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%, sequenceidentity to SEQ ID NO: 3. Another particular preferred embodimentconcerns a protease variant comprising one or more of the substitutionsselected from the group consisting of S171{W, K, E, N}; S173P; S175 {A,V, P} or G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of SEQ ID NO: 3,wherein the variant has at least 70% identity to SEQ ID NO: 3 such as atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%,e.g.at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least99.5%, at least 99.6, but less than 100%, sequence identity to the SEQID NO: 3. An even more preferred embodiment concerns a protease variantcomprising one or both of the substitutions 173P and/or S175P of SEQ IDNO: 3, wherein the variant has at least 70% identity to SEQ ID NO: 3such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%,e.g.at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least99.5%, at least 99.6, but less than 100%, sequence identity to the SEQID NO: 3.

In one aspect, the protease variant comprises a substitution at position171, in a preferred aspect the variant comprises a substitution atposition 171 with W, K, E or N, in another preferred aspect, the variantcomprises an W at position 171, in yet another preferred aspect, thevariant comprises the substitution S171W, wherein the parent is themature polypeptide with SEQ ID NO: 3. In another preferred aspect, thevariant comprises an K at position 171, in yet another preferred aspect,the variant comprises the substitution S171K, wherein the parent is themature polypeptide with SEQ ID NO: 3. In another preferred aspect, thevariant comprises an E at position 171, in yet another preferred aspect,the variant comprises the substitution S171E, wherein the parent is themature polypeptide with SEQ ID NO: 3. In another preferred aspect, thevariant comprises an N at position 171, in yet another preferred aspect,the variant comprises the substitution S171N, wherein the parent is themature polypeptide with SEQ ID NO: 3

In a further aspect, the variant comprises a substitution at position171 with W, K, E or N wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotherpreferred aspect, the variant comprises an W at position 171, in yetanother preferred aspect, the variant comprises the substitution S171W,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, e.g. at least 99.1%,at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6, but less than 100%. In another preferred aspect, the variantcomprises an K at position 171, in yet another preferred aspect, thevariant comprises the substitution S171K, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%. In another preferred aspect, the variant comprises an E atposition 171, in yet another preferred aspect, the variant comprises thesubstitution S171E, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotherpreferred aspect, the variant comprises an N at position 171, in yetanother preferred aspect, the variant comprises the substitution S171N,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, e.g. at least 99.1%,at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6, but less than 100%. In an even further aspect, the variantcomprises a substitution at position 171 with W, K, E or N wherein thevariant has at least 70% sequence identity to SEQ ID NO: 3, such as atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%,e.g. at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6,but less than 100%, and having an increased stability relative to aprotease with SEQ ID NO: 3 or a protease parent having the identicalamino acid sequence of said variant but not having the substitutions atone or more of said positions, when tested in Example 2 as describedunder “Material and Methods”. In another preferred aspect, the variantcomprises a W at position 171, in yet another preferred aspect, thevariant comprises the substitution S171W, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%,e.g. at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6, but less than100%, and having an increased stability relative to a protease with SEQID NO: 3 or a protease parent having the identical amino acid sequenceof said variant but not having the substitutions at one or more of saidpositions, when tested in the Example 2 as described under “Material andMethods”. In another preferred aspect, the variant comprises an K atposition 171, in yet another preferred aspect, the variant comprises thesubstitution S171K, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%,e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%,at least 99.5%, at least 99.6, but less than 100%, and having anincreased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in the Example 2 as described under “Material and Methods”. Inanother preferred aspect, the variant comprises an E at position 171, inyet another preferred aspect, the variant comprises the substitutionS171E, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises a N at position 171,in yet another preferred aspect, the variant comprises the substitutionS171N, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%,e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In one aspect, the protease variant comprises a substitution at position173, in a preferred aspect the variant comprises a substitution atposition 173 with P in another preferred aspect, the variant comprisesthe substitution S173P, wherein the parent is polypeptide with SEQ IDNO: 3 In another preferred aspect, the variant comprises an P atposition 173, in yet another preferred aspect, the variant comprises thesubstitution S173P, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In an evenfurther aspect, the variant comprises a substitution at position 173with P, in yet another preferred aspect, the variant comprises thesubstitution S173P, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%, and having anincreased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in Example 2 as described under “Material and Methods”.

In one aspect, the protease variant comprises a substitution at position175, in a preferred aspect the variant comprises a substitution atposition 175 with A, V or P, in another preferred aspect, the variantcomprises an A at position 175, in yet another preferred aspect, thevariant comprises the substitution S175A, wherein the parent is apolypeptide with SEQ ID NO: 3. In another preferred aspect, the variantcomprises an V at position 175, in yet another preferred aspect, thevariant comprises the substitution S175V, wherein the parent is themature polypeptide with SEQ ID NO: 3. In another preferred aspect, thevariant comprises an P at position 175, in yet another preferred aspect,the variant comprises the substitution S175P, wherein the parent is apolypeptide with SEQ ID NO: 3.

In a further aspect, the variant comprises a substitution at position175 with A, V or P wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotherpreferred aspect, the variant comprises an A at position 175, in yetanother preferred aspect, the variant comprises the substitution S175A,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%,e.g. at least 99.1%, atleast 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6, but less than 100%. In another preferred aspect, the variantcomprises an V at position 175, in yet another preferred aspect, thevariant comprises the substitution S175V, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%. In another preferred aspect, the variant comprises an P atposition 175, in yet another preferred aspect, the variant comprises thesubstitution S175P, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In an evenfurther aspect, the variant comprises a substitution at position 175with A, V or P wherein the variant has at least 70% sequence identity toSEQ ID NO: 3, such as at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%, e.g. atleast 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least99.5%, at least 99.6, but less than 100%, and having an increasedstability relative to a protease with SEQ ID NO: 3 or a protease parenthaving the identical amino acid sequence of said variant but not havingthe substitutions at one or more of said positions, when tested inExample 2 as described under “Material and Methods”. In anotherpreferred aspect, the variant comprises an A at position 175, in yetanother preferred aspect, the variant comprises the substitution S175A,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, e.g. at least 99.1%,at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6, but less than 100%, and having an increased stability relative toa protease with SEQ ID NO: 3 or a protease parent having the identicalamino acid sequence of said variant but not having the substitutions atone or more of said positions, when tested in the Example 2 as describedunder “Material and Methods”. In another preferred aspect, the variantcomprises an V at position 175, in yet another preferred aspect, thevariant comprises the substitution S175V, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in the Example 2 as described under“Material and Methods”. In another preferred aspect, the variantcomprises an P at position 175, in yet another preferred aspect, thevariant comprises the substitution S175P, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in the Example 2 as described under“Material and Methods”

In another preferred aspect, the variant comprises a C at position 179,in yet another preferred aspect, the variant comprises the substitutionG179C, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with C, in yet another preferred aspect, the variant comprises thesubstitution G179C, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotheraspect, the variant comprises a substitution at position 179 with C, inyet another preferred aspect, the variant comprises the substitutionG179C, wherein the variant has at least 70% sequence identity to SEQ IDNO: 2, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises a V at position 179,in yet another preferred aspect, the variant comprises the substitutionG179V, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with V, in yet another preferred aspect, the variant comprises thesubstitution G179V, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotherpreferred aspect, the variant comprises a substitution at position 179with V, in yet another preferred aspect, the variant comprises thesubstitution G179V, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%, and having anincreased stability relative to a protease with SEQ ID NO: 3 or aprotease parent having the identical amino acid sequence of said variantbut not having the substitutions at one or more of said positions, whentested in the Example 2 as described under “Material and Methods”.

In another preferred aspect, the variant comprises a Q at position 179,in yet another preferred aspect, the variant comprises the substitutionG179Q, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with Q, in yet another preferred aspect, the variant comprises thesubstitution G179Q, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a anotheraspect, the variant comprises a substitution at position 179 with Q, inyet another preferred aspect, the variant comprises the substitutionG179Q, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”.

In another preferred aspect, the variant comprises an S at position 179,in yet another preferred aspect, the variant comprises the substitutionG179S, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with S, in yet another preferred aspect, the variant comprises thesubstitution G179, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In a another aspect, the variant comprises asubstitution at position 179 with S, in yet another preferred aspect,the variant comprises the substitution G179, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in the Example 2 as described under“Material and Methods”.

In another preferred aspect, the variant comprises an T at position 179,in yet another preferred aspect, the variant comprises the substitutionG179T, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with T, in yet another preferred aspect, the variant comprises thesubstitution G179T, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In anotheraspect, the variant comprises a substitution at position 179 with T, inyet another preferred aspect, the variant comprises the substitutionG179T, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”.

In another preferred aspect, the variant comprises an E at position 179,in yet another preferred aspect, the variant comprises the substitutionG179E, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with E, in yet another preferred aspect, the variant comprises thesubstitution G179E, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a anotheraspect, the variant comprises a substitution at position 179 with E, inyet another preferred aspect, the variant comprises the substitutionG179E, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”.

In another preferred aspect, the variant comprises an H at position 179,in yet another preferred aspect, the variant comprises the substitutionG179H, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with H, in yet another preferred aspect, the variant comprises thesubstitution G179H, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a anotheraspect, the variant comprises a substitution at position 179 with H, inyet another preferred aspect, the variant comprises the substitutionG179H, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods” In another preferredaspect, the variant comprises a K at position 179, in yet anotherpreferred aspect, the variant comprises the substitution G179K, whereinthe parent is a polypeptide with SEQ ID NO: 3. In a further aspect, thevariant comprises a substitution at position 179 with K, in yet anotherpreferred aspect, the variant comprises the substitution G179K, whereinthe variant has at least 70% sequence identity to SEQ ID NO: 3, such asat least 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6,but less than 100%. In a another aspect, the variant comprises asubstitution at position 179 with K, in yet another preferred aspect,the variant comprises the substitution G179K, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in the Example 2 as described under“Material and Methods”.

In another preferred aspect, the variant comprises an M at position 179,in yet another preferred aspect, the variant comprises the substitutionG179M, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with M, in yet another preferred aspect, the variant comprises thesubstitution G179M, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%. In another aspect, the variant comprises asubstitution at position 179 with M, in yet another preferred aspect,the variant comprises the substitution G179M, wherein the variant has atleast 70% sequence identity to SEQ ID NO: 3, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in the Example 2 as described under“Material and Methods”.

In another preferred aspect, the variant comprises an N at position 179,in yet another preferred aspect, the variant comprises the substitutionG179N, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with N, in yet another preferred aspect, the variant comprises thesubstitution G179N, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a furtheraspect, the variant comprises a substitution at position 179 with N, inyet another preferred aspect, the variant comprises the substitutionG179N, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”.

In another preferred aspect, the variant comprises an A at position 179,in yet another preferred aspect, the variant comprises the substitutionG179A, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with A, in yet another preferred aspect, the variant comprises thesubstitution G179H, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a anotheraspect, the variant comprises a substitution at position 179 with A, inyet another preferred aspect, the variant comprises the substitutionG179A, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In another preferred aspect, the variant comprises an Y at position 179,in yet another preferred aspect, the variant comprises the substitutionG179Y, wherein the parent is a polypeptide with SEQ ID NO: 3. In afurther aspect, the variant comprises a substitution at position 179with Y, in yet another preferred aspect, the variant comprises thesubstitution G179Y, wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 3, such as at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6, but less than 100%. In a anotheraspect, the variant comprises a substitution at position 179 with Y, inyet another preferred aspect, the variant comprises the substitutionG179Y, wherein the variant has at least 70% sequence identity to SEQ IDNO: 3, such as at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, e.g. at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6, but less than 100%, and having an increased stabilityrelative to a protease with SEQ ID NO: 3 or a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions, when tested in theExample 2 as described under “Material and Methods”

In one aspect, the protease variant further comprises a substitution atposition 180, in a preferred aspect the variant further comprises asubstitution at position 180 with Y in another preferred aspect, thevariant further comprises the substitution F180Y, wherein the parent isa polypeptide with SEQ ID NO: 3. In a further aspect, the variantcomprises a substitution at position 180 with Y, in yet anotherpreferred aspect, the variant further comprises the substitution F180Y,wherein the variant has at least 70% sequence identity to SEQ ID NO: 3,such as at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, e.g. at least 99.1%,at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6, but less than 100%. In another preferred aspect, the variantcomprises an Y at position 180, in yet another preferred aspect, thevariant further comprises the substitution F180Y, wherein the varianthas at least 70% sequence identity to SEQ ID NO: 3, such as at least71%, at least 72%, at least 73%, at least 74%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but lessthan 100%, and having an increased stability relative to a protease withSEQ ID NO: 3 or a protease parent having the identical amino acidsequence of said variant but not having the substitutions at one or moreof said positions, when tested in Example 2 as described under “Materialand Methods”.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171 and 173, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171 and 175, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171 and 179, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171 and 180, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173 and 175, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173 and 179, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173 and 180, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 175 and 179, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 175 and 180, such as those described above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, and 175, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, and 179, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, and 180, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 175, and 179, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 175, and 180, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 179, and 180, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173, 175, and 179, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173, 175, and 180, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 175, 179, and 180, such as those describedabove.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, 175, and 179, such as thosedescribed above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, 175, and 180, such as thosedescribed above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 173, 175, 179 and 180, such as thosedescribed above.

In another aspect, the variant comprises substitutions at positionscorresponding to positions 171, 173, 175, 179 and 180, such as thosedescribed above.

In another aspect, the variant comprises one or more (several)substitutions selected from the group consisting of S171 {W, K, E, N},S173 {P}, S175 {A, V, P} or G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}.In another aspect the variant mentioned above further comprise thesubstitution F180Y.

In another aspect, the variant comprises the substitutions S171W+S173Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171K+S173Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171E+S173Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171N+S173Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171W+S175Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171K+S175Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171E+S175Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171N+S175Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171W+S175Vof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171K+S175Vof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171E+S175Vof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171N+S175Vof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171W+S175Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171K+S175Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171E+S175Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171N+S175Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171W+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S171K+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S171E+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S171N+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S171W+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171K+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171E+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S171N+F180Yof the polypeptide with SEQ ID NO: 3

In another aspect, the variant comprises the substitutions S173P+S175Aof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S173P+S175Vof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S173P+S175Pof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S173P+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S173P+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S175A+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S175V+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S175P+G179{C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutions S175A+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S175V+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S175P+F180Yof the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions G179 {C, V,Q, S, T, E, H, K, M, N, A, Y}+F180Y of the polypeptide with SEQ ID NO:3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175V of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175P of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175V of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175P of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175V of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175P of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175A of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175V of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175P of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of the polypeptidewith SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175A+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175V+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS173P+S175P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutions S175A+G179{C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of the polypeptide with SEQID NO: 3.

In another aspect, the variant comprises the substitutions S175V+G179{C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of the polypeptide with SEQID NO: 3.

In another aspect, the variant comprises the substitutions S175P+G179{C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of the polypeptide with SEQID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y} of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175V+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175V+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175V+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175V+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175P+F180Y of the polypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175A+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175V+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171W+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171K+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171E+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

In another aspect, the variant comprises the substitutionsS171N+S173P+S175P+G179 {C, V, Q, S, T, E, H, K, M, N, A, Y}+F180Y of thepolypeptide with SEQ ID NO: 3.

The variant may further comprise a substitution at one or more (several)other positions. For example, the variants may comprise an alteration ata position corresponding to positions selected from the group consistingof 39, 40, 70, 74, 81, 102, 121, 132, 137, 144, 155, 159, 162, 174, 176;177, 241, 247, 256, 274, 286, 297 of SEQ ID NO 3. In one embodiment thevariant of the invention comprises one of the following substitutions:Y39D; T40{D,P}; Q70N; T74M; L81{F,H,V}; A102T; 1121{V,T}; G132 {I,E};1137{M;E}; S144{Q,R}; D155N; G159S; V162R; G174{S,T}; N176G; T177S;T241P; I247M; H256F; S274I; V286Q; T297P wherein each positioncorrespond to the positions of SEQ ID NO: 3.

In another aspect, a variant according to the invention comprises asubstitution at one or more (e.g., several) positions corresponding topositions 171, 173, 175 or 179. In another aspect, a variant accordingto the invention comprises a substitution at two positions correspondingto any of positions 171, 173, 175, 179 and 180. In another aspect, avariant according to the invention comprises a substitution at threepositions corresponding to any of positions 171, 173, 175, 179 and 180.In another aspect, a variant according to the invention comprises asubstitution at four positions corresponding to any of positions 171,173, 175, 179 and 180. In another aspect, a variant according to theinvention comprises a substitution at each position corresponding topositions 171, 173, 175, 179 and 180.

The variants may further comprise one or more additional alterations atone or more (e.g., several) other positions. The amino acid changes maybe of a minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of 1-30 amino acids; smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Asn/Gln, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Glu/Gln, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

For example, the variants may comprise a substitution at a positioncorresponding to any of the positions 171, 173, 175 or 179 and furthercomprises an alteration at any of the positions selected from the groupconsisting of positions 39, 40, 70, 74, 81, 102, 121, 132, 137, 144,155, 159, 162, 174, 176; 177, 180, 241, 247, 256, 274, 286, 297. In apreferred embodiment the alteration at any of the positions selectedfrom the group consisting of 39, 40, 70, 74, 81, 102, 121, 132, 137,144, 155, 159, 162, 174, 176; 177, 180 241, 247, 256, 274, 286, 297 is asubstitution. In a particular preferred embodiment the variantsaccording to the invention comprises any of the following substitutionsS171 {W, K, E, N}, S173P, S175 {A, V, P} or G179 {C, V, Q, S, T, E, H,K, M, N, A, Y} of SEQ ID NO: 3, wherein the variant further comprisesone or more substitution selected from the group consisting of: Y39D;T40{D,P}; Q70N; T74M; L81{F,H,V}; A102T; I121{V,T}; G132 {I,E};1137{M;E}; S144{Q,R}; D155N; G159S; V162R; G174{S,T}; N176G; T177S;F180Y; T241P; I247M; H256F; S274I; V286Q; T297P.

In one embodiment of the invention, the variants according to theinvention comprise any of the following substitutions compared to SEQ IDNO 3:

S171N S175P I121V S175P L81V S175P A102T S175P I137E S175P S175A G179SI121V S175A L81F S175A L81H S175A L81V S175A A102T S175A I137E S175AS144Q S175P S144Q S175A S144R G179Q I121T S175P S144R S171N S173P S274II137M S173P S171N S173P L81F S173P L81H S173P A102T S173P S144Q S173PS144R F180Y I137E S173P S173P S175P F180Y S173Y G174S S175A F180Y S173PG174T S175V T177S F180Y S173P G174K S175P N176G T177S F180Y S173P T241PG179S F180Y G183A A187V S173Y G174S S175A F180Y T241P D155N G159S S173YG174S S175A F180Y S173Y G174S S175A F180Y S274I S173Y G174S S175A F180YV286Q T40D S173Y G174S S175A F180Y V162R S173P G174T S175V T177S F180YI121V S173P G174T S175V T177S F180Y L81V S173P G174T S175V T177S F180YA102T S173P G174T S175V T177S F180Y S173P G174T S175V T177S F180Y T241PI137E S173P G174T S175V T177S F180Y S144Q S173P G174T S175V T177S F180YQ70N S173P G174T S175V T177S F180Y D155N G159S S173P G174T S175V T177SF180Y S173P G174T S175V T177S F180Y S274I S173P G174T S175V T177S F180YV286Q T40D S173P G174T S175V T177S F180Y S171N S173P G174T S175V T177SF180Y V162R S173P G174K S175P N176G T177S F180Y I121V S173P G174K S175PN176G T177S F180Y L81V S173P G174K S175P N176G T177S F180Y A102T S173PG174K S175P N176G T177S F180Y S173P G174K S175P N176G T177S F180Y T241PI137E S173P G174K S175P N176G T177S F180Y S144Q S173P G174K S175P N176GT177S F180Y Q70N S173P G174K S175P N176G T177S F180Y D155N G159S S173PG174K S175P N176G T177S F180Y S173P G174K S175P N176G T177S F180Y S274IS173P G174K S175P N176G T177S F180Y V286Q T40D S173P G174K S175P N176GT177S F180Y S171N S173P G174K S175P N176G T177S F180Y I121V I137E S173YG174S S175A F180Y L81V I137E S173Y G174S S175A F180Y I137E S173Y G174SS175A F180Y T241P Q70N I137E S173Y G174S S175A F180Y I137E S173Y G174SS175A F180Y S274I I137E S173Y G174S S175A F180Y T297P I121V I137E S173PG174T S175V T177S F180Y L81V I137E S173P G174T S175V T177S F180Y I137ES173P G174T S175V T177S F180Y T241P Q70N I137E S173P G174T S175V T177SF180Y I137E S173P G174T S175V T177S F180Y V286Q I137E S173P G174T S175VT177S F180Y T297P I137E S171N S173P G174T S175V T177S F180Y I137E S173PG174T S175A T177S F180Y I121V I137E S173P G174K S175P N176G T177S F180YQ70N I137E S173P G174K S175P N176G T177S F180Y I137E S173P G174K S175PN176G T177S F180Y S274I I137E S173P G174K S175P N176G T177S F180Y V286QI137E S173P G174K S175P N176G T177S F180Y T297P I137E S171N S173P G174KS175P N176G T177S F180Y I137E S173P G174K S175A N176G T177S F180Y V162RS173Y G174S S175A F180Y A102T S173Y G174S S175A F180Y G132I S173P G174TS175V T177S F180Y S173P G174T S175V T177S F180Y T297P S173P G174T S175AT177S F180Y S173P S175A I137E S144Q S173Y G174S S175A F180Y T40L I137ES173Y G174S S175A F180Y I137E S173Y S175A F180Y I137E S173P S175V T177SF180Y I137E S173P S175P N176G T177S F180Y S171N S173Y G174S S175A F180YS173P G174K S175A N176G T177S F180Y I137E S173Y G174S S175A F180Y V286QI137E S173P G174T S175V T177S F180Y S274I T74M I137E S173P T79I I137ES173P L81G S173P L34I I137E S173P Y39D I137E S173P T40P I137E S173PI137E S173P I247M I137E S173P H256F I137E S144Q S173P G174T S175V T177SF180Y I137E S144Q S173P G174K S175P N176G T177S F180Y S173Y G174S S175PF180Y I137E S173Y G174S S175P F180Y S173P G174T S175P T177S F180Y I137ES173P G174T S175P T177S F180Y I137E S173P G174T S175P T177S F180Y T297PV162R S171N S173P G174K S175P N176G T177S F180Y I137E S171N S173P G174KS175P N176G T177S F180Y T241P I137E S171N S173P G174K S175P N176G T177SF180Y T297P I137E S173P S175P F180Y V162R S173P S175P F180Y S173P S175PF180Y S274I S173P S175P F180Y T241P S173P S175P F180Y T297P S173P S175PF180Y V286Q I137E S173Y G174S S175A F180Y S144Q S173Y G174S S175A F180YQ70N S173Y G174S S175A F180Y I121V S173Y G174S S175A F180Y L81V S173YG174S S175A F180Y G132I S173P G174K S175P N176G T177S F180Y S173P G174KS175P N176G T177S F180Y T297P

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide. ForTY-145 (SEQ ID NO: 3) the catalytic triad comprising the amino acidsD35, H72 and S251 is essential for protease activity of the enzyme.

In an embodiment, the variant has improved catalytic activity comparedto the parent enzyme.

In an embodiment, the variant has improved stability compared to theparent enzyme or compared to a protease having the identical amino acidsequence of said variant but not having a substitution at one or more ofsaid specified positions or compared to a protease with SEQ ID NO: 3,wherein stability is measured in Example 2 as described in “Material andMethods” herein.

In an embodiment, a variant according to the invention has improvedstability compared to the parent enzyme or compared to a protease havingthe identical amino acid sequence of said variant but not having asubstitution at one or more of said specified positions or compared to aprotease with SEQ ID NO: 3.

Parent Proteases Protease Variant

The term “variant” and the term “protease variant” are defined above.

Homologous Protease Sequences

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the artto identify suitable homologous proteases, which can be modifiedaccording to the invention.

Substantially homologous parent protease variants may have one or more(several) amino acid substitutions, deletions and/or insertions, in thepresent context the term “one or more” is used interchangeably with theterm “several”. These changes are preferably of a minor nature, that isconservative amino acid substitutions as described above and othersubstitutions that do not significantly affect the three-dimensionalfolding or activity of the protein or polypeptide; small deletions,typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification (an affinity tag), such asa poly-histidine tract, or protein A (Nilsson et al., 1985, EMBO J. 4:1075; Nilsson et al., 1991, Methods Enzymol. 198: 3. See, also, ingeneral, Ford et al., 1991, Protein Expression and Purification 2:95-107.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

The parent protease may comprise or consist of the amino acid sequenceof SEQ ID NO: 3 or an allelic variant thereof; or a fragment thereofhaving protease activity. In one aspect, the parent protease comprisesor consists of the amino acid sequence of SEQ ID NO: 3.

The parent protease may be (a) a polypeptide having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 3; (b) apolypeptide encoded by a polynucleotide that hybridizes under medium orhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii); or (c) a polypeptide encoded by a polynucleotide having atleast 70% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1.

In an aspect, the parent protease has a sequence identity to thepolypeptide with SEQ ID NO: 3 of at least 70%, such as at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76% atleast 77% at least 78% at least 79% at least 80%, at least 81% at least82% at least 83% at least 84% at least 85%, at least 86% at least 87% atleast 88% at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94% at least 95% identity, at least 96%, at least97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6, or 100%,which have protease activity.

In one aspect, the amino acid sequence of the parent protease differs byno more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, fromthe mature polypeptide with SEQ ID NO: 3.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3. In another aspect, the parent comprises orconsists of amino acids 1 to 311 of SEQ ID NO: 2.

In another aspect, the parent protease is encoded by a polynucleotidethat hybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, or high stringency conditions,or very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii), (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 3 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) a sequence encoding the mature polypeptide of SEQ ID NO:2; (iv) the full-length complement thereof; or (v) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleotide acid probeis a 80 to 1140 nucleotides long fragment of SEQ ID NO: 1, e.g. 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long.In another aspect, the nucleic acid probe is a polynucleotide thatencodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof;or a fragment thereof. In another aspect, the nucleic acid probe is SEQID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2 ofat least 71%, at least 72%, at least 73%, at least 74%, at least 70%,e.g., at least 75%, at least 76%, at least 77% at least 78% at least 79%at least 80%, at least 81% at least 82% at least 83% at least 84% atleast 85%, at least 86% at least 87% at least 88% at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%,e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, atleast 99.5%, at least 99.6 or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention.

A fusion polypeptide is produced by fusing a polynucleotide encodinganother polypeptide to a polynucleotide of the present invention.Techniques for producing fusion polypeptides are known in the art, andinclude ligating the coding sequences encoding the polypeptides so thatthey are in frame and that expression of the fusion polypeptide is undercontrol of the same promoter(s) and terminator. Fusion polypeptides mayalso be constructed using intein technology in which fusion polypeptidesare created post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from organisms of any genus. For purposes ofthe present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the parent encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, theparent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease

In one aspect, the parent is a Bacillus sp. protease, e.g., the proteasewith SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO 2.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving protease activity, comprising: (a) introducing into a parentprotease a substitution at one or more (e.g., several) positionscorresponding to positions 171, 173, 175 or 179 of SEQ ID NO: 3, whereinthe variant has protease activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Thus, the invention also relates to a method for obtaining a proteasevariant, comprising introducing into a parent protease a substitution atone or more positions corresponding to positions 171, 173, 175 or 179 ofSEQ ID NO: 3; and recovering the variant.

Another embodiment concerns a method for obtaining a protease variant,comprising substitution of one or more amino acids in the loopcorresponding to positions 171, 173, 175 or 179 of SEQ ID NO: 3,especially a method as described above, wherein the parent protease isselected from the group consisting of: a polypeptide having at least70%, such as at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76% at least 77% at least 78% at least 79% at least80%, at least 81% at least 82% at least 83% at least 84% at least 85%,at least 86% at least 87% at least 88% at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94% at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%,e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, atleast 99.5% or at least 99.6 sequence identity to SEQ ID NO: 3;

a polypeptide encoded by a polynucleotide that hybridizes under mediumor high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) a sequence encoding the maturepolypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i)or (ii);

a polypeptide encoded by a polynucleotide having at least 70% identityto the mature polypeptide coding sequence of SEQ ID NO: 1 or a sequenceencoding the mature polypeptide of SEQ ID NO: 2; and

a fragment of the mature polypeptide of SEQ ID NO: 2, which has proteaseactivity.

Thus, a particular aspect concerns a method for obtaining a proteasevariant, comprising introducing into a parent protease a substitution ofone or more amino acids in the loop corresponding to positions 171, 173,175 or 179 of SEQ ID NO: 3, wherein the substitution(s) is/are performedin SEQ ID NO: 3, and wherein the substitutions are selected from thegroup consisting of S171 {W, K, E, N}, S173 {P}, S175 {A, V, P} or G179{C, V, Q, S, T, E, H, K, M, N, A, Y}. Additionally a furthersubstitution can be introduced at position corresponding to 180 of SEQID NO: 3, especially the substitution is F180Y.

One aspect of the invention relates to methods of producing the variantsaccording to the invention, wherein the method comprises substitution ofat least one amino acid in the loop corresponding to positions 171, 173,175 or 179 of SEQ ID NO: 3, wherein (a) the variant has a sequenceidentity to SEQ ID NO: 3 of at least 70% and less than 100% and (b) thevariant has protease activity.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 171 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 173, 175, 179 and 180 of SEQ ID NO:3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 173 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 171, 175, 179 and 180 of SEQ ID NO:3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 175 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 171, 173, 179 and 180 of SEQ ID NO:3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 179 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 171, 173, 175 and 180 of SEQ ID NO:3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 180 ofSEQ ID NO: 3 and further comprises a substitution at one or morepositions corresponding to positions 171, 173, 175 and 179 of SEQ ID NO:3.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 171 ofSEQ ID NO: 3 with an amino acid selected from {W, K, E, N}.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 173 ofSEQ ID NO: 3 with P.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 175 ofSEQ ID NO: 3 with an amino acid selected from {A, V, P}.

In one embodiment, the variant produced according to said methodcomprises a substitution at a position corresponding to position 179 ofSEQ ID NO: 3 with an amino acid selected from {C, V, Q, S, T, E, H, K,M, N, A, Y}.

In one embodiment, the variant produced according to said method mayfurther comprise a substitution at a position corresponding to position180 of SEQ ID NO: 3 with Y.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rmB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

In one certain aspect, the variants according to the invention hasimproved stability in detergents compared to the parent enzyme orcompared to a protease having the identical amino acid sequence of saidvariant but not having the substitutions at one or more of saidspecified positions or compared to a protease with SEQ ID NO 3, whereinstability is measured in Example 2 as described in “Material andMethods” herein.

Besides enzymes the detergent compositions may comprise additionalcomponents. The choice of additional components is within the skill ofthe artisan and includes conventional ingredients, including theexemplary non-limiting components set forth below. The choice ofcomponents may include, for fabric care, the consideration of the typeof fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product. Although components mentioned below arecategorized by general header according to a particular functionality,this is not to be construed as a limitation, as a component may compriseadditional functionalities as will be appreciated by the skilledartisan.

Detergent Compositions of the Present Invention

The variants of the present invention may be added to a detergentcomposition in an amount corresponding to 0.001-100 mg of protein, suchas 0.01-100 mg of protein, preferably 0.005-50 mg of protein, morepreferably 0.01-25 mg of protein, even more preferably 0.05-10 mg ofprotein, most preferably 0.05-5 mg of protein, and even most preferably0.01-1 mg of protein per liter of wash liquor.

The variants of the present invention may be stabilized usingstabilizing agents, which may be selected from the group containingpropylene glycol, glycerol, a sugar, a sugar alcohol, lactic acid, boricacid, borate and phenyl boronic acid derivates, such as those where theresidue R in the phenyl boronic acid derivative is a C₁-C₆ alkyl groupand among these, more preferably, CH₃, CH₃CH₂ or CH₃CH₂CH₂. The residueR in the phenyl boronic acid derivative may also be hydrogen. Oneexample of a phenyl boronic acid derivative is 4-formylphenylboronicacid (4-FPBA) with the following formula:

Phenyl boronic acid derivatives can furthermore have other chemicalmodifications on the phenyl ring, and in particular they can contain oneor more methyl, amino, nitro, chloro, fluoro, bromo, hydroxyl, formyl,ethyl, acetyl, t-butyl, anisyl, benzyl, trifluoroacetyl,N-hydroxysuccinimide, t-butyloxycarbonyl, benzoyl, 4-methylbenzyl,thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl,benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulfenyl,4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl,2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl,2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl residues or groups orcombinations thereof. All stabilizing agents can be present in thedetergent composition in all protonated or deprotonated forms.Furthermore, all such compounds, in particular their deprotonated forms,can be associated with cations. Preferred cations in this respect aremonovalent or polyvalent, in particular divalent, cations, in particularNa ions (Na⁺), K ions (K⁺), Li ions (Li⁺), Ca ions (Ca²⁺), Mg ions(Mg²⁺), Mn ions (Mn²⁺) and Zn ions (Zn²⁺). The detergent compositionsmay comprise two or more stabilizing agents e.g. such as those selectedfrom the group consisting of propylene glycol, glycerol, 4-formylphenylboronic acid and borate. One example is a detergent compositioncomprising 4-formylphenyl boronic acid and/or borate. The phenyl boronicacid derivative may be contained in the detergent composition in aquantity of from 0.00001 to 5.0 wt %, preferably from 0.0001 to 3.0 wt%, from 0.001 to 2.0 wt %, from 0.005 to 1.0 wt %, from 0.01 to 0.5 wt%, from 0.02 to 0.3 wt % Preferably, the boric acid/borate is containedin a quantity of from 0.001 to 5.5 wt. % and increasingly preferably offrom 0.01 to 4.5 wt. %, from 0.05 to 3.5 and from 0.1 to 3, 0.4 bis2.49, 0.5 bis 1.5 wt. % in the detergent composition. Addition of acombination of borate and 4-formylphenyl boronic acid has been found tobe particularly effective, leading to a high increase in enzymestability in detergent compositions. Preferably, the boric acid/borateis contained in a quantity of from 0.001 to 5.5 wt. % and increasinglypreferably from 0.075 to 4.5 wt. %, from 0.09 to 3.5 and from 0.1 to2.49 wt. %, and the phenyl boronic acid derivative is contained in aquantity of from 0.001 to 0.08 wt. % and increasingly preferably from0.003 to 0.06 wt. %, from 0.005 to 0.05 wt. %, from 0.007 to 0.03 wt. %and from 0.009 to 0.01 wt. % in a detergent composition. Particularlypreferred is the addition of 4-formylphenyl boronic acid in an amount of1.0 to 2.0 wt % in combination with 1.0 wt % borate.

The variants according to the invention may also be stabilized usingpeptide aldehydes or ketones such as described in WO 2005/105826 and WO2009/118375. A variant of the present invention may also be incorporatedin the detergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 1%to about 40% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylehanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, and combinationsthereof, Alkyl quaternary ammonium compounds, Alkoxylated quaternaryammonium (AQA),

When included therein the detergent will usually contain from about 0.2%to about 40% by weight of a non-ionic surfactant, for example from about0.5% to about 30%, in particular from about 1% to about 20%, from about3% to about 10%, such as from about 3% to about 5%, or from about 8% toabout 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually contain from about 1%to about 40% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually contain from about 1%to about 40% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, and sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluenesulfonates (STS), sodium xylene sulfonates (SXS), sodium cumenesulfonates (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 50% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry, ADW and hard surfaces cleaning detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The detergent composition may also contain 0-65% by weight, such asabout 5% to about 40%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO 09/102854, U.S. Pat. No. 5,977,053.

Bleaching Systems

The detergent may contain 0-10% by weight, such as about 1% to about 5%,of a bleaching system. Any bleaching system known in the art for use inlaundry, ADW and hard surfaces cleaning detergents may be utilized.Suitable bleaching system components include bleaching catalysts,photobleaches, bleach activators, sources of hydrogen peroxide such assodium percarbonate and sodium perborates, preformed peracids andmixtures thereof. Suitable preformed peracids include, but are notlimited to, peroxycarboxylic acids and salts, percarbonic acids andsalts, perimidic acids and salts, peroxymonosulfuric acids and salts,for example, Oxone (R), and mixtures thereof. Non-limiting examples ofbleaching systems include peroxide-based bleaching systems, which maycomprise, for example, an inorganic salt, including alkali metal saltssuch as sodium salts of perborate (usually mono- or tetra-hydrate),percarbonate, persulfate, perphosphate, persilicate salts, incombination with a peracid-forming bleach activator. By bleach activatoris meant herein a compound which reacts with peroxygen bleach likehydrogen peroxide to form a peracid. The peracid thus formed constitutesthe activated bleach. Suitable bleach activators to be used hereininclude those belonging to the class of esters amides, imides oranhydrides. Suitable examples are tetracetyl ethylene diamine (TAED),sodium 3,5,5 trimethyl hexanoyloxybenzene sulphonat, diperoxy dodecanoicacid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS),tetraacetylethylenediamine (TAED) and 4-(nonanoyloxy)benzenesulfonate(NOBS), and/or those disclosed in WO98/17767. A particular family ofbleach activators of interest was disclosed in EP624154 and particularlypreferred in that family is acetyl triethyl citrate (ATC). ATC or ashort chain triglyceride like Triacin has the advantage that it isenvironmental friendly as it eventually degrades into citric acid andalcohol. Furthermore acethyl triethyl citrate and triacetin has a goodhydrolytical stability in the product upon storage and it is anefficient bleach activator. Finally ATC provides a good buildingcapacity to the laundry additive. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthaloylamino)percapronic acid (PAP). The bleaching system may alsoinclude a bleach catalyst. In some embodiments the bleach component maybe an organic catalyst selected from the group consisting of organiccatalysts having the following formulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO2007/087258, WO2007/087244, WO2007/087259, WO2007/087242. Suitablephotobleaches may for example be sulfonated zinc phthalocyanine

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of polyethyleneterephthalate and polyoxyethene terephthalate (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridin-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions may also include fabric hueing agents such asdyes or pigments which when formulated in detergent compositions candeposit onto a fabric when said fabric is contacted with a wash liquorcomprising said detergent compositions thus altering the tint of saidfabric through absorption/reflection of visible light. Fluorescentwhitening agents emit at least some visible light. In contrast, fabrichueing agents alter the tint of a surface as they absorb at least aportion of the visible light spectrum. Suitable fabric hueing agentsinclude dyes and dye-clay conjugates, and may also include pigments.Suitable dyes include small molecule dyes and polymeric dyes. Suitablesmall molecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Colour Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, forexample as described in WO2005/03274, WO2005/03275, WO2005/03276 andEP1876226 (hereby incorporated by reference). A detergent compositionpreferably comprises from about 0.00003 wt % to about 0.2 wt %, fromabout 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % toabout 0.04 wt % fabric hueing agent. The composition may comprise from0.0001 wt % to 0.2 wt % fabric hueing agent, this may be especiallypreferred when the composition is in the form of a unit dose pouch.Suitable hueing agents are also disclosed in, e.g., WO 2007/087257,WO2007/087243.

(Additional) Enzymes

In one embodiment, the variants according to the invention are combinedwith one or more enzymes, such as at least two enzymes, more preferredat least three, four or five enzymes. Preferably, the enzymes havedifferent substrate specificity, e.g., proteolytic activity, amylolyticactivity, lipolytic activity, hemicellulytic activity or pectolyticactivity.

The detergent additive as well as the detergent composition may compriseone or more additional enzymes such as carbohydrate-active enzymes likecarbohydrase, pectinase, mannanase, amylase, cellulase, arabinase,galactanase, xylanase, or protease, lipase, a, cutinase, oxidase, e.g.,a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andWO99/001544.

Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence ofat least 97% identity to the amino acid sequence of position 1 toposition 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44xyloglucanase, which a xyloglucanase enzyme having a sequence of atleast 60% identity to positions 40-559 of SEQ ID NO: 2 of WO2001/062903.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S) Carezyme Premium™ (Novozymes A/S), Celluclean™(Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™(Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Mannanases

Suitable mannanases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. The mannanasemay be an alkaline mannanase of Family 5 or 26. It may be a wild-typefrom Bacillus or Humicola, particularly B. agaradhaerens, B.licheniformis, B. halodurans, B. clausii, or H. insolens. Suitablemannanases are described in WO 1999/064619. A commercially availablemannanase is Mannaway (Novozymes A/S).

Proteases:

Suitable proteases include those of bacterial, fungal, plant, viral oranimal origin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellulomonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130,160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235,236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred theprotease variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R,*36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A,V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D,Y167A, R170S, A194P, G195E, V199M, V2051, L217D, N218D, M222S, A232V,K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Preferenz™, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®,Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Eraser®,Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.),BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variantshereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases:

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the 554V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases:

Suitable amylases which can be used together with the variants of theinvention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/023873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions:K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459,D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are thosehaving the substitution in one of more of the following positions:K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T,G476K and G477K and/or deletion in position R178 and/or S179 or of T180and/or G181. Most preferred amylase variants of SEQ ID NO: 1 are thosehaving the substitutions:

E187P+I203Y+G476K

E187P+I203Y+R458N+T459S+D460T+G476K

wherein the variants optionally further comprises a substitution atposition 241 and/or a deletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675or variants having 90% sequence identity to SEQ ID NO: 1 thereof.Preferred variants of SEQ ID NO: 1 are those having a substitution, adeletion or an insertion in one of more of the following positions: N21,D97, V128 K177, R179, S180, 1181, G182, M200, L204, E242, G477 and G478.More preferred variants of SEQ ID NO: 1 are those having thesubstitution in one of more of the following positions: N21D, D97N,V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion inposition R179 and/or S180 or of I181 and/or G182. Most preferred amylasevariants of SEQ ID NO: 1 are those having the substitutions:

N21D+D97N+V128I

wherein the variants optionally further comprises a substitution atposition 200 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes A/S), and Rapidase™ Purastar™/Effectenz™, Powerase, PreferenzS1000, Preferenz S100 and Preferenz S110 (from Genencor InternationalInc./DuPont).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

Other Enzymes:

A protease variant according to the invention may also be combined withadditional enzymes such as pectate lyases e.g. Pectawash™,chlorophyllases etc. The protease variant of the invention may be mixedwith any additional enzyme.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e., a separate additive or a combined additive, can be formulated, forexample, as a granulate, liquid, slurry, etc. Preferred detergentadditive formulations are granulates, in particular non-dustinggranulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants The detergent compositions can also contain dispersants. Inparticular powdered detergents may comprise dispersants. Suitablewater-soluble organic materials include the homo- or co-polymeric acidsor their salts, in which the polycarboxylic acid comprises at least twocarboxyl radicals separated from each other by not more than two carbonatoms. Suitable dispersants are for example described in PowderedDetergents, Surfactant science series volume 71, Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents—The detergent compositions may alsoinclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. When present in a subjectcomposition, the dye transfer inhibiting agents may be present at levelsfrom about 0.0001% to about 10%, from about 0.01% to about 5% or evenfrom about 0.1% to about 3% by weight of the composition.

Fluorescent whitening agent—A detergent compositions will preferablyalso contain additional components that may tint articles being cleaned,such as fluorescent whitening agent or optical brighteners. Wherepresent the brightener is preferably at a level of about 0.01% to about0.5%. Any fluorescent whitening agent suitable for use in a laundrydetergent composition may be used in the composition. The most commonlyused fluorescent whitening agents are those belonging to the classes ofdiaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivativesand bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulphonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use include the 1-3-diaryl pyrazolines and the7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

Soil release polymers—The detergent composition may also include one ormore soil release polymers which aid the removal of soils from fabricssuch as cotton and polyester based fabrics, in particular the removal ofhydrophobic soils from polyester based fabrics. The soil releasepolymers may for example be nonionic or anionic terephthalte basedpolymers, polyvinyl caprolactam and related copolymers, vinyl graftcopolymers, polyester polyamides see for example Chapter 7 in PowderedDetergents, Surfactant science series volume 71, Marcel Dekker, Inc.Another type of soil release polymers are amphiphilic alkoxylated greasecleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore random graft co-polymers are suitable soilrelease polymers Suitable graft co-polymers are described in more detailin WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 2003/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

Anti-redeposition agents—The detergent compositions may also include oneor more anti-redeposition agents such as carboxymethylcellulose (CMC),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethyleneand/or polyethyleneglycol (PEG), homopolymers of acrylic acid,copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,structurants for liquid detergents and/or structure elasticizing agents.

Formulation of Detergent Products

The detergent composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a regular orcompact powder, a granule, a paste, a gel, or a regular, compact orconcentrated liquid.

Detergent formulation forms: Layers (same or different phases), Pouches,versus forms for Machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxyprpyl methyl cellulose (HPMC). Preferably the level of polymer inthe film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blend compositions comprising hydrolytically degradableand water soluble polymer blends such as polyactide and polyvinylalcohol (known under the Trade reference M8630 as sold by Chris CraftIn. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethyleneglycerol, Propylene glycol, sorbitol and mixtures thereof. The pouchescan comprise a solid laundry cleaning composition or part componentsand/or a liquid cleaning composition or part components separated by thewater soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. Ref:(US2009/0011970 A1)

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

Definition/Characteristics of the Forms:

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO09/092699,EP1705241, EP1382668, WO07/001262, U.S. Pat. No. 6,472,364, WO04/074419or WO09/102854. Other useful detergent formulations are described inWO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342,WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296,WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125,WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375,WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545,WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391,WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770,WO09/021813, WO09/030632, and WO09/015951.

WO2011025615, WO2011016958, WO2011005803, WO2011005623, WO2011005730,WO2011005844, WO2011005904, WO2011005630, WO2011005830, WO2011005912,WO2011005905, WO2011005910, WO2011005813, WO2010135238, WO2010120863,WO2010108002, WO2010111365, WO2010108000, WO2010107635, WO2010090915,WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979,WO2010030540, WO2010030541, WO2010030539, WO2010024467, WO2010024469,WO2010024470, WO2010025161, WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051, WO2010102861, WO2010099997,WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957,WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO2011023716, WO2010142539, WO2010118959, WO2010115813, WO2010105942,WO2010105961, WO2010105962, WO2010094356, WO2010084203, WO2010078979,WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486,WO2010066631, WO2010066632, WO2010063689, WO2010060821, WO2010049187,WO2010031607, WO2010000636,

Methods and Uses

The protease variants of the present invention may be added to and thusbecome a component of a detergent composition, wherein said variantcomprises a substitution of one or more amino acids in the loopcorresponding to positions 171, 173, 175 or 179 of SEQ ID NO: 3 andwherein the variant has at least 70%, such as at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76% at least 77%at least 78% at least 79% at least 80%, at least 81% at least 82% atleast 83% at least 84% at least 85%, at least 86% at least 87% at least88% at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94% at least 95% identity, at least 96%, at least 97%, atleast 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5% or at least 99.6% sequenceidentity to SEQ ID NO: 3. Detergent compositions is generally used incleaning processes such as laundry and/or hard surface cleaning e.g.dish wash. A detergent compositions may comprise at least one a variantwherein said variant comprises one or more of the followingsubstitutions S171 {W, K, E, N}, S173 {P}, S175 {A, V, P} or G179 {C, V,Q, S, T, E, H, K, M, N, A, Y} of SEQ ID NO: 3, wherein the variant has asequence identity to SEQ ID NO: 3 of at least 70% such as at least 71%,at least 72%, at least 73%, at least 74%, at least 75%, at least 76% atleast 77% at least 78% at least 79% at least 80%, at least 81% at least82% at least 83% at least 84% at least 85%, at least 86% at least 87% atleast 88% at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94% at least 95% identity, at least 96%, at least97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5% or at least 99.6 sequenceidentity to SEQ ID NO: 3 and the variant has protease activity. The atleast one protease variant preferably has increased detergent stabilityrelative to the parent or relative to a protease parent having theidentical amino acid sequence of said variant but not having thesubstitutions at one or more of said positions when tested in theExample 2, as described under “Material and Methods”.

Additionally, any of the protease variants described above may alsocontain a substitution at position F180Y.

A detergent composition may be formulated, for example, as a hand ormachine laundry detergent composition including a laundry additivecomposition suitable for pre-treatment of stained fabrics and a rinseadded fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

A cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. A process for laundering of fabricsand/or garments may be a process comprises treating fabrics with awashing solution containing a detergent composition, and at least oneprotease variant. A cleaning process or a textile care process can forexample be carried out in a machine washing process or in a manualwashing process. The washing solution can for example be an aqueouswashing solution containing a detergent composition.

The fabrics and/or garments subjected to a washing, cleaning or textilecare process may be conventional washable laundry, for example householdlaundry. Preferably, the major part of the laundry is garments andfabrics, including knits, woven, denims, non-woven, felts, yarns, andtowelling. The fabrics may be cellulose based such as naturalcellulosics, including cotton, flax, linen, jute, ramie, sisal or coiror manmade cellulosics (e.g., originating from wood pulp) includingviscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell orblends thereof. The fabrics may also be non-cellulose based such asnatural polyamides including wool, camel, cashmere, mohair, rabbit andsilk or synthetic polymer such as nylon, aramid, polyester, acrylic,polypropylen and spandex/elastane, or blends thereof as well as blend ofcellulose based and non-cellulose based fibers. Examples of blends areblends of cotton and/or rayon/viscose with one or more companionmaterial such as wool, synthetic fibers (e.g., polyamide fibers, acrylicfibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloridefibers, polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g., rayon/viscose, ramie, flax, linen,jute, cellulose acetate fibers, lyocell).

The last few years there has been an increasing interest in replacingcomponents in detergents, which is derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change new enzyme activities or new enzymes havingalternative and/or improved properties compared to the common useddetergent enzymes such as proteases, lipases and amylases is needed toachieve a similar or improved wash performance when compared to thetraditional detergent compositions.

Proteases and variants hereof are usable in proteinaceous stain removingprocesses. The proteinaceous stains may be stains such as food stains,e.g., baby food, sebum, cocoa, egg, blood, milk, ink, grass, or acombination hereof.

Typical detergent compositions include various components in addition tothe enzymes, these components have different effects, some componentslike the surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away, other components like bleach systems remove discolor oftenby oxidation and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Yet other components likebuilder and chelator softens, e.g., the wash water by removing the metalions form the liquid.

The enzyme compositions may further comprise at least one or more of thefollowing: a surfactant, a builder, a chelator or chelating agent,bleach system or bleach component in laundry or dish wash.

The amount of a surfactant, a builder, a chelator or chelating agent,bleach system and/or bleach component may be reduced compared to amountof surfactant, builder, chelator or chelating agent, bleach systemand/or bleach component used without the added protease variant of theinvention. Preferably the at least one component which is a surfactant,a builder, a chelator or chelating agent, bleach system and/or bleachcomponent is present in an amount that is 1% less, such as 2% less, suchas 3% less, such as 4% less, such as 5% less, such as 6% less, such as7% less, such as 8% less, such as 9% less, such as 10% less, such as 15%less, such as 20% less, such as 25% less, such as 30% less, such as 35%less, such as 40% less, such as 45% less, such as 50% less than theamount of the component in the system without the addition of proteasevariants of the invention, such as a conventional amount of suchcomponent. Detergent compositions may also be composition which is freeof at least one component which is a surfactant, a builder, a chelatoror chelating agent, bleach system or bleach component and/or polymer.

Washing Method

Detergent compositions are ideally suited for use in laundryapplications. These methods include a method for laundering a fabric.The method comprises the steps of contacting a fabric to be launderedwith a cleaning laundry solution comprising a detergent composition. Thefabric may comprise any fabric capable of being laundered in normalconsumer use conditions. The solution preferably has a pH from about 5.5to about 11.5. The compositions may be employed at concentrations fromabout 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. Thewater temperatures typically range from about 5° C. to about 95° C.,including about 10° C., about 15° C., about 20° C., about 25° C., about30° C., about 35° C., about 40° C., about 45° C., about 50° C., about55° C., about 60° C., about 65° C., about 70° C., about 75° C., about80° C., about 85° C. and about 90° C. The water to fabric ratio istypically from about 1:1 to about 30:1.

In particular embodiments, the washing method is conducted at a pH fromabout 5.0 to about 11.5, or from about 6 to about 10.5, about 5 to about11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 toabout 9, about 5.5 to about 8, about 5.5. to about 7, about 6 to about11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 toabout 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about11, about 7 to about 10, about 7 to about 9, or about 7 to about 8,about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 toabout 11, about 9 to about 10, about 10 to about 11, preferably about5.5 to about 11.5.

In particular embodiments, the washing method is conducted at a degreeof hardness of from about 0° dH to about 30° dH, such as about 1° dH,about 2° dH, about 3° dH, about 4° dH, about 5° dH, about 6° dH, about7° dH, about 8° dH, about 9° dH, about 10° dH, about 11° dH, about 12°dH, about 13° dH, about 14° dH, about 15° dH, about 16° dH, about 17°dH, about 18° dH, about 19° dH, about 20° dH, about 21° dH, about 22°dH, about 23° dH, about 24° dH, about 25° dH, about 26° dH, about 27°dH, about 28° dH, about 29° dH, about 30° dH. Under typical Europeanwash conditions, the degree of hardness is about 16° dH, under typicalUS wash conditions about 6° dH, and under typical Asian wash conditions,about 3° dH.

The compositions for use in the methods described above may furthercomprises at least one additional enzyme as set forth in the “otherenzymes” section above, such as an enzyme selected from the group ofhydrolases such as proteases, lipases and cutinases, carbohydrases suchas amylases, cellulases, hemicellulases, xylanases, and pectinase or acombination hereof.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Automatic Mechanical Stress Assay (AMSA)for Laundry

In order to assess the wash performance in laundry washing experimentsare performed, using the Automatic Mechanical Stress Assay (AMSA). Withthe AMSA, the wash performance of a large quantity of small volumeenzyme-detergent solutions can be examined. The AMSA plate has a numberof slots for test solutions and a lid firmly squeezing the laundrysample, the textile to be washed against all the slot openings. Duringthe washing time, the plate, test solutions, textile and lid arevigorously shaken to bring the test solution in contact with the textileand apply mechanical stress in a regular, periodic oscillating manner.For further description see WO02/42740 especially the paragraph “Specialmethod embodiments” at page 23-24.

The wash performance is measured as the brightness of the colour of thetextile washed. Brightness can also be expressed as the intensity of thelight reflected from the sample when illuminated with white light. Whenthe sample is stained the intensity of the reflected light is lower,than that of a clean sample. Therefore the intensity of the reflectedlight can be used to measure wash performance.

Colour measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak, Midtager 29, DK-2605 Brøndby, Denmark), which is used tocapture an image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image are converted into values for red,green and blue (RGB). The intensity value (Int) is calculated by addingthe RGB values together as vectors and then taking the length of theresulting vector:

Int=√{square root over (r ² +g ² +b ²)}.

TABLE 1 Composition of model detergents and test materials Modeldetergent and test materials were as follows: Laundry liquid 0.3 to 0.5%xanthan gum, model detergent 0.2 to 0.4% antifoaming agent, 6 to 7%glycerol, 0.3 to 0.5% ethanol, 4 to 7% FAEOS (fatty alcohol ethersulfate), 24 to 28% nonionic surfactants, 1% boric acid, 1 to 2% sodiumcitrate (dihydrate), 2 to 4% soda, 14 to 16% coconut fatty acid, 0.5%HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0 to 0.4% PVP(polyvinylpyrrolidone), 0 to 0.05% optical brighteners, 0 to 0.001% dye,remainder deionized water. Test material PC-03 (Chocolate-milk/ink oncotton/polyester) C-05 (Blood/milk/ink on cotton)

General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Protease Activity Assays: 1) Suc-AAPF-pNA Activity Assay:

The proteolytic activity can be determined by a method employing theSuc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and itis a blocked peptide which can be cleaved by endo-proteases. Followingcleavage a free PNA molecule is liberated and it has a yellow colour andthus can be measured by visible spectrophotometry at wavelength 405 nm.The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400,dissolved in DMSO).

The protease sample to be analyzed was diluted in residual activitybuffer (100 mM Tris pH8.6). The assay was performed by transferring 60μl of diluted enzyme samples to 96 well microtiter plate and adding 140μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). Thesolution was mixed at room temperature and absorption is measured every20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the protease in question under the given set of conditions.The protease sample should be diluted to a level where the slope islinear.

Example 1 Preparation and Testing of Protease Variants Preparation andExpression of Variants

Site-directed variants were constructed of the TY145 protease (SEQ IDNO: 3) comprising specific insertions/deletions/substitutions in the 170to 180 region on the N-terminal side according to the invention. Thevariants were made by traditional cloning of DNA fragments (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, 1989) using PCR together with properly designed mutagenicoligonucleotides that introduced the desired mutations in the resultingsequence. Mutagenic oligos were synthesized corresponding to the DNAsequence flanking the desired site(s) of mutation, separated by the DNAbase pairs defining the insertions/deletions/substitutions. In thismanner, the variants listed in table 2a below were constructed andproduced.

Fermentation of Variants

Fermentation may be performed by methods well known in the art or asfollows. A B. subtilis strain harboring the relevant expression plasmidwas streaked on a LB-agar plate with a relevant antibiotic (6 μg/mlchloramphenicol), and grown overnight at 37° C.

The colonies were transferred to 100 ml PS-1 media supplemented with therelevant antibiotic in a 500 ml shaking flask containing a rich media(e.g. PS-1: 100 g/L Sucrose (Danisco cat. no. 109-0429), 40 g/L crustsoy (soy bean flour), 10 g/L Na₂HPO₄.12H₂O (Merck cat. no. 6579), 0.1ml/L Pluronic PE 6100 (BASF 102-3098)). Cultivation typically takes 4days at 30° C. shaking with 220 rpm. Cells and other undissolvedmaterial were removed from the fermentation broth by centrifugation at4500 rpm for 20-25 minutes. Afterwards the supernatant was filtered toobtain a clear solution.

Example 2

In this example, the above-described PNA-Suc-AAPF assay is used todetermine the residual protease activity after incubation in thepresence of liquid detergent. In general the residual protease activitywas determined after incubation in liquid detergent (final concentrationof 90%) at the indicated temperatures and incubation times and theactivity is then compared to the activity of a unstressed incubated at4° C. For the determination of protease stability in detergent theenzymes to be tested were adjusted to a concentration of 0.15 mg/ml ofenzyme protein by dilution in enzyme dilution buffer (100 mM Tris pH8.6, 0.0225% (w/V) Brij-35, 2 mM CaCl₂). 30 μl of the protease solutionand 270 μl liquid detergent (Model liquid detergent) were transferred toa 96 well microtiter plate (Nunc 96U PP) in 4 replicates. One smallmagnet (5×2 mm) was placed in each well, and the blend was mixed for 30minutes at room temperature on a magnetic stirrer. After mixing 20 μl istransferred to new 96 well microtiter plate and incubated at 4° C. for24 hours (unstressed sample). Heat seal with alu-foil carefullymicrotiter plate and incubate at indicated temperature for 24 hours(stressed samples). After incubation, the samples on the plates wereanalyzed for protease activity as described in the PNA-Suc-AAPF Assayfor determination of residual protease activity. It should be noted,that in order to reduce interference from other detergent ingredientsthan the enzyme on the assay, both unstressed and stressed samples werediluted to the same protein concentration. After incubation, withdraw 20μl of stressed samples and add 150 μl residual activity buffer (100 mMTris pH8.6), and mix using magnetic stirrer for 5 minutes. Transfer 60μl of diluted sample to new 96 well microtiter plate. Before use preparePNA-Suc-AAPF substrate working solution in residual activity buffer(0.72 mg/ml in 100 mM Tris pH8.6). Add 140 μl substrate working solutionto diluted sample, mix and measure immediately absorbance at 405 nm for5-10 minutes every 20 seconds at room temperature. Use Vmax only fromlinear range of kinetic curves. Repeat residual activity measurement forunstressed sample by adding 150 μl residual activity buffer (100 mM TrispH8.6) to microtiter plate with 20 μl unstressed sample (incubated at 4°C.), and mix using magnetic stirrer for 5 minutes. Transfer 60 μl ofdiluted sample to new 96 well microtiter plate. Add 140 μl substrateworking solution to diluted sample, mix and measure immediatelyabsorbance at 405 nm for 5-10 minutes every 20 seconds at roomtemperature. Use Vmax only from linear range of kinetic curves. It wasensured in all experiments that the reference protease was included atleast once on all test microtiter plates.

The residual activity (% RA) was calculated as % RA=100*Vmax (stressedsample)/Vmax (unstressed sample).

The half-life (T½ (h)) is calculated: T½ (hours)=T (hours)*LN(0.5)/LN(%RA/100) with T being incubation time (hours) and % RA is residualactivity.

TABLE 3a stability of variants measured at 35° C. Half-life T½ (hours)(incubation Mutations at 35° C. for 24 h in 90% detergent) TY-145 (SEQID NO 3) 18 G179C 36 G179V 56 G179Q 65 G179S 29 G179Y 67 G179T 31 G179E28 G179H 79 G179K 47 G179M 29 G179A 82 G179N 37 S171W 28 S171K 23 S171E23 S175A 44 S175V 30 S175P 108

TABLE 3b stability of variants measured at 35° C. Half-life T½ (hours)(incubation at Mutations 35° C. for 24 h in 90% detergent) TY-145 (SEQID NO 3) 18 I121V S175P 96 S175A G179S 78 L81V S175A 90 A102T S175A 86S144R G179Q 96 S144R S171N 96 S144R F180Y 70 S171N 40 G179S F180Y G183AA187V 95

TABLE 4a Stability of variants measured at 42° C. Half-life T½ (hours)(incubation Mutations at 42° C. for 24 h in 90% detergent) TY-145 (SEQID NO 3) 0 S173P 96 S171N S175P 10 I121V S175P 7 L81V S175P 6 A102TS175P 6 I137E S175P 7 1121V S175A 3 L81F S175A 3 I137E S175A 4 S144QS175P 6 S144Q S175A 3 I121T S175P 8

TABLE 4b Stability of variants measured at 42° C. Half-life T½ (hours)(incubation at 42° C. for 24 h in Mutations 90% detergent) TY-145 (SEQID NO 3) 0 S173Y G174S S175A F180Y 96 I137E S173Y G174S S175A F180Y 11S144Q S173Y G174S S175A F180Y 9 S173Y G174S S175A F180Y S274I 10 S173YG174S S175A F180Y V286Q 10 T40D S173Y G174S S175A F180Y 9 S173P G174KS175P N176G T177S F180Y 30 D155N G159S S173Y G174S S175A F180Y 6 I121VS173P G174K S175P N176G T177S F180Y 52 L81V S173P G174K S175P N176GT177S F180Y 41 A102T S173P G174K S175P N176G T177S F180Y 37 S173P G174KS175P N176G T177S F180Y T241P 48 I137E S173P G174K S175P N176G T177SF180Y 41 S144Q S173P G174K S175P N176G T177S F180Y 41 Q70N S173P G174KS175P N176G T177S F180Y 59 S173P G174K S175P N176G T177S F180Y V286Q 45T40D S173P G174K S175P N176G T177S F180Y 32 S171N S173P G174K S175PN176G T177S F180Y 52 S173P G174K S175P N176G T177S F180Y S274I 44

TABLE 5a Stability of variants measured at 47° C. Half-life T½ (hours)(incubation at 47° C. Mutations for 24 h in 90% detergent) TY-145 (SEQID NO 3) 0 S173P 26 S171N S175P 6 I121V S175P 6 L81V S175P 6 A102T S175P6 I137E S175P 6 I121V S175A 6 L81F S175A 6 I137E S175A 6 S144Q S175P 6S144Q S175A 6 I121T S175P 6 S173P S274I 29 I137M S173P 30 S171N S173P 38L81F S173P 28 A102T S173P 26 S144Q S173P 29 I137E S173P 35 S173P T241P32 S173P S175A 34 L81G S173P 39

TABLE 5b Stability of variants measured at 47° C. Half-life T½ (hours)(incubation at 47° C. for Mutations 24 h in 90% detergent) TY-145 (SEQID NO 3) 0 S173P S175P F180Y 96 T74M I137E S173P 26 T79I I137E S173P 27L34I I137E S173P 36 Y39D I137E S173P 39 T40P I137E S173P 46 I137E S173PI247M 41 I137E S173P H256F 31 S173Y G174S S175A F180Y 22 I137E S173YS175A F180Y 6 S173Y G174S S175P F180Y 6 I137E S173P S175P F180Y 96 V162RS173P S175P F180Y 96 S173P S175P F180Y S274I 96 S173P G174T S175V T177SF180Y 39 S173Y G174S S175A F180Y T241P 6 I137E S173Y G174S S175A F180Y 6S144Q S173Y G174S S175A F180Y 6 Q70N S173Y G174S S175A F180Y 6 S173YG174S S175A F180Y S274I 6 S173Y G174S S175A F180Y V286Q 6 T40D S173YG174S S175A F180Y 6 V162R S173Y G174S S175A F180Y 6 I121V S173Y G174SS175A F180Y 6 L81V S173Y G174S S175A F180Y 6 A102T S173Y G174S S175AF180Y 6 S173P G174T S175A T177S F180Y 39 I137E S173P S175V T177S F180Y34 S171N S173Y G174S S175A F180Y 6 I137E S173Y G174S S175P F180Y 7 S173PG174T S175P T177S F180Y 61 S173P G174K S175P N176G T177S F180Y 6 V162RS173P G174T S175V T177S F180Y 68 I121V S173P G174T S175V T177S F180Y 49L81V S173P G174T S175V T177S F180Y 41 A102T S173P G174T S175V T177SF180Y 42 S173P G174T S175V T177S F180Y T241P 61 I137E S173P G174T S175VT177S F180Y 79 S144Q S173P G174T S175V T177S F180Y 65 Q70N S173P G174TS175V T177S F180Y 80 S173P G174T S175V T177S F180Y S274I 56 S173P G174TS175V T177S F180Y V286Q 56 T40D S173P G174T S175V T177S F180Y 37 S171NS173P G174T S175V T177S F180Y 58 I121V I137E S173Y G174S S175A F180Y 6L81V I137E S173Y G174S S175A F180Y 6 I137E S173Y G174S S175A F180Y T241P6 Q70N I137E S173Y G174S S175A F180Y 6 I137E S173Y G174S S175A F180YS274I 6 I137E S173Y G174S S175A F180Y T297P 8 G132I S173P G174T S175VT177S F180Y 45 S173P G174T S175V T177S F180Y T297P 72 I137E S144Q S173YG174S S175A F180Y 6 T40L I137E S173Y G174S S175A F180Y 6 I137E S173PS175P N176G T177S F180Y 6 S173P G174K S175A N176G T177S F180Y 6 I137ES173Y G174S S175A F180Y V286Q 6 I137E S173P G174T S175P T177S F180Y 88D155N G159S S173P G174T S175V T177S F180Y 17 V162R S173P G174K S175PN176G T177S F180Y 11 I121V S173P G174K S175P N176G T177S F180Y 8 L81VS173P G174K S175P N176G T177S F180Y 7 A102T S173P G174K S175P N176GT177S F180Y 7 S173P G174K S175P N176G T177S F180Y T241P 9 I137E S173PG174K S175P N176G T177S F180Y 9 S144Q S173P G174K S175P N176G T177SF180Y 7 Q70N S173P G174K S175P N176G T177S F180Y 8 S173P G174K S175PN176G T177S F180Y V286Q 9 T40D S173P G174K S175P N176G T177S F180Y 7S171N S173P G174K S175P N176G T177S F180Y 9 I121V I137E S173P G174TS175V T177S F180Y 73 I137E S173P G174T S175V T177S F180Y T241P 90 I137ES173P G174T S175V T177S F180Y V286Q 75 I137E S173P G174T S175V T177SF180Y T297P 96 I137E S171N S173P G174T S175V T177S F180Y 74 I137E S173PG174K S175A N176G T177S F180Y 6 G132I S173P G174K S175P N176G T177SF180Y 6 S173P G174K S175P N176G T177S F180Y T297P 16 I137E S173P G174TS175V T177S F180Y S274I 64 I137E S144Q S173P G174T S175V T177S F180Y 56S173P G174K S175P N176G T177S F180Y S274I 7 I121V I137E S173P G174KS175P N176G T177S F180Y 13 Q70N I137E S173P G174K S175P N176G T177SF180Y 13 I137E S173P G174K S175P N176G T177S F180Y S274I 12 I137E S173PG174K S175P N176G T177S F180Y V286Q 12 I137E S173P G174K S175P N176GT177S F180Y T297P 23 I137E S171N S173P G174K S175P N176G T177S F180Y 15I137E S144Q S173P G174K S175P N176G T177S F180Y 11 V162R S171N S173PG174K S175P N176G T177S F180Y 16 I137E S171N S173P G174K S175P N176GT177S F180Y T241P 22 I137E S171N S173P G174K S175P N176G T177S F180YT297P 41

TABLE 6 Stability of variants measured at 52° C. Half-life T½ (hours)(incubation at 52° C. for 24 h in Mutations 90% detergent) TY-145 (SEQID NO 3) 0 I137E S173P 6 S173P S175P F180Y 27 T40P I137E S173P 7 I137ES173P S175P F180Y 39 V162R S173P S175P F180Y 45 S173P S175P F180Y S274I30 S173P S175P F180Y T241P 31 S173P S175P F180Y V286Q 30 S173P S175PF180Y T297P 46 S173P G174T S175V T177S F180Y 11 V162R S173P G174T S175VT177S F180Y 16 I137E S173P G174T S175V T177S F180Y 15 Q70N S173P G174TS175V T177S F180Y 16 S173P G174T S175V T177S F180Y V286Q 14 S171N S173PG174T S175V T177S F180Y 15 I137E S173P G174T S175A T177S F180Y 14 S173PG174T S175V T177S F180Y T297P 20 I137E S173P G174T S175P T177S F180Y 37L81V I137E S173P G174T S175V T177S F180Y 17 Q70N I137E S173P G174T S175VT177S F180Y 26 I137E S173P G174T S175V T177S F180Y V286Q 16 I137E S171NS173P G174T S175V T177S F180Y 23 I137E S144Q S173P G174T S175V T177SF180Y 15 I137E S173P G174T S175P T177S F180Y T297P 96

Example 3

The wash performance of protease variants were tested using a liquiddetergent on 3 different technical stains using the Automatic MechanicalStress Assay.

The experiments were conducted as described in the AMSA for laundrymethod using a single cycle wash procedure, with the detergentcomposition and swatches described in table 1 and the experimentalconditions as specified in table 7 below.

TABLE 7 Experimental conditions for AMSA for tables 7a to 7f Testsolution 4.66 g/L liquid model detergent Test solution volume 160 microL pH As is Wash time 20 minutes Temperature 20° C. Water hardness 16° dHProtease concentration 0 (blank), 10 nM or 30 nM Swatch PC-03, C-05Water hardness was adjusted to 16° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:CO₃ ²⁻)=5:1:6 for PC-03 and 5:1:11 for C-05 to thetest system. After washing the textiles were flushed in tap water anddried.

TABLE 7a Wash performance of single and double protease variants on aChocolate, Milk, Soot stain, reference enzyme is TY-145 (SEQ ID NO 3)Wash performance relative to SEQ ID NO 3, at Mutations 20 C. on PC-03G179S 96 S173P 108 I121V S175A 106 L81V S175A 97 S144Q S175P 112 S144QS175A 97 S173P S274I 102 L81F S173P 103 S144Q S173P 101 I137E S173P 97S173P T241P 111 S173P S175A 106

TABLE 7b Wash performance of protease variants on a Chocolate, Milk,Soot stain, reference enzyme is TY-145 (SEQ ID NO 3) Wash performancerelative to SEQ ID NO 3, at 20 C. Mutations on PC-03 S173P S175P F180Y91 S173Y G174S S175A F180Y 101 S173P G174T S175V T177S F180Y 98 I137ES173Y G174S S175A F180Y 108 S144Q S173Y G174S S175A F180Y 101 S173YG174S S175A F180Y S274I 117 S173Y G174S S175A F180Y V286Q 94 V162R S173YG174S S175A F180Y 121 I121V S173Y G174S S175A F180Y 110 S173P G174TS175A T177S F180Y 106 S173P G174K S175P N176G T177S F180Y 98 D155N G159SS173Y G174S S175A F180Y 118 V162R S173P G174T S175V T177S F180Y 125I121V S173P G174T S175V T177S F180Y 99 L81V S173P G174T S175V T177SF180Y 98 I137E S173P G174T S175V T177S F180Y 112 S173P G174T S175V T177SF180Y V286Q 94 S173P G174T S175V T177S F180Y T297P 94 S173P G174K S175AN176G T177S F180Y 107 D155N G159S S173P G174T S175V T177S F180Y 116V162R S173P G174K S175P N176G T177S F180Y 135 I121V S173P G174K S175PN176G T177S F180Y 117 L81V S173P G174K S175P N176G T177S F180Y 104 A102TS173P G174K S175P N176G T177S F180Y 110 I137E S173P G174K S175P N176GT177S F180Y 122 S144Q S173P G174K S175P N176G T177S F180Y 107 S173PG174K S175P N176G T177S F180Y V286Q 112 S171N S173P G174K S175P N176GT177S F180Y 125 I137E S173P G174T S175V T177S F180Y V286Q 100 I137ES173P G174K S175A N176G T177S F180Y 106 S173P G174K S175P N176G T177SF180Y T297P 117 D155N G159S S173P G174K S175P N176G T177S 99 F180Y S173PG174K S175P N176G T177S F180Y S274I 125 I121V I137E S173P G174K S175PN176G T177S F180Y 189 I137E S173P G174K S175P N176G T177S F180Y S274I 97I137E S173P G174K S175P N176G T177S F180Y 96 V286Q I137E S173P G174KS175P N176G T177S F180Y T297P 128 I137E S171N S173P G174K S175P N176GT177S 121 F180Y

TABLE 7c Wash performance of single and double protease variants on aBlood, Milk, Ink stain, reference enzyme is TY-145 (SEQ ID NO 3) Washperformance relative to SEQ ID NO 3, at Mutations 20 C. on C-05 S173P110 I121V S175P 113 L81V S175P 105 A102T S175P 100 S175A G179S 95 I121VS175A 111 L81V S175A 101 I137E S175A 95 S144Q S175P 116 S144Q S175A 104I121T S175P 98 S144R S171N 103 S173P S274I 96 I137M S173P 105 S171NS173P 115 L81F S173P 111 L81H S173P 103 A102T S173P 117 S144Q S173P 108I137E S173P 120 S173P T241P 129 S173P S175A 100

TABLE 7d Wash performance of protease variants on a Blood, Milk, Inkstain, reference enzyme is TY-145 (SEQ ID NO 3) Wash performancerelative to SEQ ID NO 3, at 20 C. Mutations on C-05 S173P S175P F180Y 98S173Y G174S S175A F180Y 120 I137E S173Y S175A F180Y 103 S173P G174TS175V T177S F180Y 106 I137E S173Y G174S S175A F180Y 108 S144Q S173YG174S S175A F180Y 98 Q70N S173Y G174S S175A F180Y 105 S173Y G174S S175AF180Y S274I 95 S173Y G174S S175A F180Y V286Q 112 V162R S173Y G174S S175AF180Y 116 I121V S173Y G174S S175A F180Y 123 L81V S173Y G174S S175A F180Y116 A102T S173Y G174S S175A F180Y 101 S173P G174T S175A T177S F180Y 111S171N S173Y G174S S175A F180Y 103 S173P G174K S175P N176G T177S F180Y107 D155N G159S S173Y G174S S175A F180Y 97 V162R S173P G174T S175V T177SF180Y 124 I121V S173P G174T S175V T177S F180Y 109 L81V S173P G174T S175VT177S F180Y 102 I137E S173P G174T S175V T177S F180Y 104 S144Q S173PG174T S175V T177S F180Y 95 Q70N S173P G174T S175V T177S F180Y 98 S173PG174T S175V T177S F180Y S274I 116 S173P G174T S175V T177S F180Y V286Q 98S171N S173P G174T S175V T177S F180Y 101 L81V I137E S173Y G174S S175AF180Y 97 I137E S173Y G174S S175A F180Y S274I 109 I137E S173Y G174S S175AF180Y T297P 100 I137E S173P G174T S175A T177S F180Y 104 S173P G174TS175V T177S F180Y T297P 113 T40L I137E S173Y G174S S175A F180Y 96 S173PG174K S175A N176G T177S F180Y 112 I137E S173Y G174S S175A F180Y V286Q 98D155N G159S S173P G174T S175V T177S F180Y 107 V162R S173P G174K S175PN176G T177S F180Y 113 I121V S173P G174K S175P N176G T177S F180Y 116 L81VS173P G174K S175P N176G T177S F180Y 114 A102T S173P G174K S175P N176GT177S F180Y 106 S173P G174K S175P N176G T177S F180Y T241P 94 I137E S173PG174K S175P N176G T177S F180Y 122 S144Q S173P G174K S175P N176G T177SF180Y 109 S173P G174K S175P N176G T177S F180Y V286Q 99 T40D S173P G174KS175P N176G T177S F180Y 105 S171N S173P G174K S175P N176G T177S F180Y116 L81V I137E S173P G174T S175V T177S F180Y 100 Q70N I137E S173P G174TS175V T177S F180Y 113 I137E S173P G174T S175V T177S F180Y V286Q 109I137E S171N S173P G174T S175V T177S F180Y 102 I137E S173P G174K S175AN176G T177S F180Y 119 G132I S173P G174K S175P N176G T177S F180Y 107S173P G174K S175P N176G T177S F180Y T297P 109 I137E S173P G174T S175VT177S F180Y S274I 99 D155N G159S S173P G174K S175P N176G T177S F180Y 115S173P G174K S175P N176G T177S F180Y S274I 106 I121V I137E S173P G174KS175P N176G T177S F180Y 153 I137E S173P G174K S175P N176G T177S F180YS274I 100 I137E S173P G174K S175P N176G T177S F180Y V286Q 113 I137ES173P G174K S175P N176G T177S F180Y T297P 126 I137E S171N S173P G174KS175P N176G T177S F180Y 122

Example 4

The wash performance of variants in detergents was determined by usingthe following standardized stains:

A: blood, milk, ink on cotton: product no. C-05 obtainable from CFT(Center for Testmaterials) B.V., Vlaardingen, Netherlands,

B: groundnut oil, pigment, high milk on cotton: product no. C-10obtainable from CFT (Center for Testmaterials) B.V., Vlaardingen,Netherlands,

C: grass on cotton: product no. E164 obtainable from EidgenössischeMaterial- and Prüfanstalt (EMPA) Testmaterialien AG [Federal materialsand testing agency, Testmaterials], St. Gallen, Switzerland.

A liquid washing agent with the following composition was used as baseformulation (all values in weight percent): 0 to 0.5% xanthan gum, 0.2to 0.4% antifoaming agent, 6 to 7% glycerol, 0.3 to 0.5% ethanol, 0 to7% FAEOS (fatty alcohol ether sulfate), 10 to 28% nonionic surfactants,0.5-1% boric acid, 1 to 2% sodium citrate (dihydrate), 2 to 4% soda, 0to 16% coconut fatty acid, 0.5% HEDP (1-hydroxyethane-(1,1-diphosphonicacid)), 0 to 0.4% PVP (polyvinylpyrrolidone), 0 to 0.05% opticalbrighteners, 0 to 0.001% dye, remainder deionized water.

Based on this base formulation, various detergents were prepared byadding respective proteases as indicated in tables 8a & b. Reference isthe protease that has the amino acid sequence described in FIG. 2 andSEQ ID NO: 3 of WO 03/055713, the reference protease already showing agood wash performance, especially in liquid detergents. The proteaseswere added in the same amounts based on total protein content (5 mg/lwash liquor).

The dosing ratio of the liquid washing agent was 4.7 grams per liter ofwashing liquor and the washing procedure was performed for 60 minutes ata temperature of 20° C. and 40° C., the water having a water hardnessbetween 15.5 and 16.5° (German degrees of hardness).

The whiteness, i.e. the brightening of the stains, was determinedphotometrically as an indication of wash performance. A Minolta CM508dspectrometer device was used, which was calibrated beforehand using awhite standard provided with the unit.

The results obtained are the difference values between the remissionunits obtained with the detergents and the remission units obtained withthe detergent containing the reference protease. A positive valuetherefore indicates an improved wash performance of the variants in thedetergent. It is evident from tables 8a (results at 40° C.) and 8b(results at 20° C.) that variants according to the invention showimproved wash performance.

TABLE 8a Wash performance of protease variants that have the same aminoacid sequence as TY-145 (SEQ ID NO: 3) except for the substitutions asper the table below on a blood, milk, ink stain on cotton (A); referenceis the protease that has the amino acid sequence described in FIG. 2 andSEQ ID NO.3 of WO 03/055713. Protease variant A I137E + S173P 2.9S173P + S175P + F180Y (variant 2) 3.2 I137E + S173P + G174T + S175V +T177S + F180Y 3.5 S173P + G174T + S175V + T177S + F180Y + S274I 2.6S173P + G174T + S175V + T177S + F180Y + T297P 3.6 S173P + S175P +F180Y + V286Q 2.5

TABLE 8b Wash performance of protease variants on a blood, milk, inkstain on cotton (A), groundnut oil, pigment, high milk on cotton (B);grass on cotton (C); reference is the protease that has the amino acidsequence described in FIG. 2 and SEQ ID NO. 3 of WO 03/055713. Proteasevariant A B C I137E + S173P 2.5 1.9 0.7 S173P + S175P + F180Y (variant2) 1.6 0.8 0.9 I137E + S173P + G174T + S175V + T177S + F180Y 2.0 2.1 0.6S173P + G174T + S175V + T177S + F180Y + S274I 1.5 2.0 1.6 S173P +G174T + S175V + T177S + F180Y + T297P 3.1 1.6 1.2 S173P + S175P +F180Y + V286Q 2.0 0.6 1.0

Example 5

The stability of variant 2 as defined in example 4 and a non-proteaseenzyme (Mannanase) was tested in the liquid detergent composition asdescribed above in Example 4 and according to the following protocol:

1. Enzyme Preparation: The correct % of the enzyme preparation is addedto the formulation and the formulation is then stored for 8 Weeks at 30°C.2. Protease activity: The enzyme activity can be measured according tomethods known in the art. Enzyme activity assays are well known to theperson skilled in the art and are routinely used. Protease activityassays are for example disclosed in Tenside, volume 7 (1970), pages125-132. The protease activity can further be determined using theSuc-AAPF-pNA activity assay which measures the release ofpara-nitroanilin (pNA) from the substratesuc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA. The release ofthe pNA causes an increase in extinction at 410 nm, of which the curveover time is a measure of enzymatic activity (cf. Del Mar et al., 1979).The measurement is conducted at temperatures of 25° C., at pH 8.6 and awavelength of 410 nm. The measuring period is 5 min. with a measuringinterval of 20 s to 60 s. The protease activity is preferably expressedin PU (protease units).3. Mannanase activity: After storage the remaining mannanase activitywas measured as described in “Assay of endo-1,4-beta-Mannanase usingBeat Annanase Tablets” (Fa. Megazyme). Enzyme samples were incubated inwith Azurine-crosslinked cardo Galactomannan in 0.2 M Tris HCl pH 8.2.The reaction was followed spectroscopically at 590 nm.4. Calculations: all activities are expressed as relative activities,either relative to the start value or relative to the normed activity ofthe control non-“diluted” granulate mix.

The stability of variant 2 was tested in the liquid detergentcomposition as described above in Example 4. To assess the effect ofborate and 4-formylphenyl boronic acid (4-FPBA) on the stability ofvariant 2, these two components were added to the liquid detergentcomposition separately or in combination in varying concentrations asper table 9a below.

TABLE 9a Stability of variant 2 in liquid detergent compositioncontaining varying concentrations of Borate and 4-formylphenyl boronicacid (4-FPBA). Without With 1% w/w Protease variant stability BorateBorate 0.9% w/w variant 2 (2% (w/w) 4-FPBA) 65% 79% 0.9% w/w variant 2(0% (w/w) 4-FPBA) 11% 80% 0.9% w/w variant 2 (1% (w/w) 4-FPBA) 57% 84%

The stability of a non-protease enzyme in the liquid detergentpreparation containing variant 2 was tested by adding 0.00001% of activeMannanase (Mannaway™ by Novozymes) to the composition.

TABLE 9b Stability of Mannanase in a liquid detergent compositioncontaining variant 2 and varying concentrations of Borate and4-formylphenyl boronic acid (4-FPBA). Without With 1% w/w Mannanasestability Borate Borate 0.9% w/w variant 2 (2% (w/w) 4-FPBA) 30% 85%0.9% w/w variant 2 (0% (w/w) 4-FPBA) 0% 81% 0.9% w/w variant 2 (1% (w/w)4-FPBA) 15% 85%

1. A protease variant, comprising a substitution at one or morepositions corresponding to positions 171, 173, 175 or 179 of SEQ ID NO:3, wherein the variant has a sequence identity to SEQ ID NO: 3 of atleast 75% and less than 100%, and the variant has protease activity. 2.The protease variant of claim 1, wherein a) the amino acid at theposition corresponding to position 171 of SEQ ID NO: 3 is selected fromthe group consisting of Trp, Lys, Glu, Asn and/or b) the amino acid atthe position corresponding to position 173 of SEQ ID NO: 3 is Pro,and/or c) the amino acid at the position corresponding to position 175of SEQ ID NO: 3 is Ala, Val, Pro, and/or d) the amino acid at theposition corresponding to position 179 of SEQ ID NO: 3 is selected fromthe group consisting of Cys, Val, Gln, Ser, Thr, Glu, His, Lys, Met,Asn, Tyr and Ala.
 3. The variant of claim 1, which comprises analteration at two positions corresponding to any of positions 171, 173,175, 179, and
 180. 4. The variant according to claim 1, wherein theamino acid at the position corresponding to position 180 of SEQ ID NO: 3is Tyr.
 5. The variant according to claim 1, wherein the amino acid atthe position corresponding to position 175 of SEQ ID NO: 3 is Pro. 6.The variant of claim 1, which further comprises one or moresubstitutions selected from the group consisting of Y39D; T40{D,P};Q70N; T74M; L81{F,H,V}; A102T; I121{V,T}; G132 {I,E}; I137{M;E};S144{Q,R}; D155N; G159S; V162R; G174{S,T}; N176G; T177S; T241P; I247M;H256F; S274I; V286Q; T297P.
 7. A protease variant, comprising any of thefollowing substitutions compared to of SEQ ID NO: 3: S171N S175P I121VS175P L81V S175P A102T S175P I137E S175P S175A G179S I121V S175A L81FS175A L81H S175A L81V S175A A102T S175A I137E S175A S144Q S175P S144QS175A S144R G179Q I121T S175P S144R S171N S173P S274I I137M S173P S171NS173P L81F S173P L81H S173P A102T S173P S144Q S173P S144R F180Y I137ES173P S173P S175P F180Y S173Y G174S S175A F180Y S173P G174T S175V T177SF180Y S173P G174K S175P N176G T177S F180Y S173P T241P G179S F180Y G183AA187V S173Y G174S S175A F180Y T241P D155N G159S S173Y G174S S175A F180YS173Y G174S S175A F180Y S274I S173Y G174S S175A F180Y V286Q T40D S173YG174S S175A F180Y V162R S173P G174T S175V T177S F180Y I121V S173P G174TS175V T177S F180Y L81V S173P G174T S175V T177S F180Y A102T S173P G174TS175V T177S F180Y S173P G174T S175V T177S F180Y T241P I137E S173P G174TS175V T177S F180Y S144Q S173P G174T S175V T177S F180Y Q70N S173P G174TS175V T177S F180Y D155N G159S S173P G174T S175V T177S F180Y S173P G174TS175V T177S F180Y S274I S173P G174T S175V T177S F180Y V286Q T40D S173PG174T S175V T177S F180Y S171N S173P G174T S175V T177S F180Y V162R S173PG174K S175P N176G T177S F180Y I121V S173P G174K S175P N176G T177S F180YL81V S173P G174K S175P N176G T177S F180Y A102T S173P G174K S175P N176GT177S F180Y S173P G174K S175P N176G T177S F180Y T241P I137E S173P G174KS175P N176G T177S F180Y S144Q S173P G174K S175P N176G T177S F180Y Q70NS173P G174K S175P N176G T177S F180Y D155N G159S S173P G174K S175P N176GT177S F180Y S173P G174K S175P N176G T177S F180Y S274I S173P G174K S175PN176G T177S F180Y V286Q T40D S173P G174K S175P N176G T177S F180Y S171NS173P G174K S175P N176G T177S F180Y I121V I137E S173Y G174S S175A F180YL81V I137E S173Y G174S S175A F180Y I137E S173Y G174S S175A F180Y T241PQ70N I137E S173Y G174S S175A F180Y I137E S173Y G174S S175A F180Y S274II137E S173Y G174S S175A F180Y T297P I121V I137E S173P G174T S175V T177SF180Y L81V I137E S173P G174T S175V T177S F180Y I137E S173P G174T S175VT177S F180Y T241P Q70N I137E S173P G174T S175V T177S F180Y I137E S173PG174T S175V T177S F180Y V286Q I137E S173P G174T S175V T177S F180Y T297PI137E S171N S173P G174T S175V T177S F180Y I137E S173P G174T S175A T177SF180Y I121V I137E S173P G174K S175P N176G T177S F180Y Q70N I137E S173PG174K S175P N176G T177S F180Y I137E S173P G174K S175P N176G T177S F180YS274I I137E S173P G174K S175P N176G T177S F180Y V286Q I137E S173P G174KS175P N176G T177S F180Y T297P I137E S171N S173P G174K S175P N176G T177SF180Y I137E S173P G174K S175A N176G T177S F180Y V162R S173Y G174S S175AF180Y A102T S173Y G174S S175A F180Y G132I S173P G174T S175V T177S F180YS173P G174T S175V T177S F180Y T297P S173P G174T S175A T177S F180Y S173PS175A I137E S144Q S173Y G174S S175A F180Y T40L I137E S173Y G174S S175AF180Y I137E S173Y S175A F180Y I137E S173P S175V T177S F180Y I137E S173PS175P N176G T177S F180Y S171N S173Y G174S S175A F180Y S173P G174K S175AN176G T177S F180Y I137E S173Y G174S S175A F180Y V286Q I137E S173P G174TS175V T177S F180Y S274I T74M I137E S173P T79I I137E S173P L81G S173PL34I I137E S173P Y39D I137E S173P T40P I137E S173P I137E S173P I247MI137E S173P H256F I137E S144Q S173P G174T S175V T177S F180Y I137E S144QS173P G174K S175P N176G T177S F180Y S173Y G174S S175P F180Y I137E S173YG174S S175P F180Y S173P G174T S175P T177S F180Y I137E S173P G174T S175PT177S F180Y I137E S173P G174T S175P T177S F180Y T297P V162R S171N S173PG174K S175P N176G T177S F180Y I137E S171N S173P G174K S175P N176G T177SF180Y T241P I137E S171N S173P G174K S175P N176G T177S F180Y T297P I137ES173P S175P F180Y V162R S173P S175P F180Y S173P S175P F180Y S274I S173PS175P F180Y T241P S173P S175P F180Y T297P S173P S175P F180Y V286Q I137ES173Y G174S S175A F180Y S144Q S173Y G174S S175A F180Y Q70N S173Y G174SS175A F180Y I121V S173Y G174S S175A F180Y L81V S173Y G174S S175A F180YG132I S173P G174K S175P N176G T177S F180Y S173P G174K S175P N176G T177SF180Y T297P


8. The variant of claim 1, which has an improved detergent stabilitycompared to the parent or compared to the protease with SEQ ID NO:
 3. 9.The variant of claim 1, wherein the variant is selected from the groupconsisting of: a) a polypeptide having at least 75% sequence identity tothe mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by apolynucleotide that hybridizes under medium, or high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or(iii) the full-length complement of (i) or (ii); c) a polypeptideencoded by a polynucleotide having at least 75% identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding themature polypeptide of SEQ ID NO: 2; and d) a fragment of the maturepolypeptide of SEQ ID NO: 2, which has protease activity.
 10. Thevariant of claim 1, wherein the variant has at least at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, e.g. at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6, but less than100%, sequence identity to the mature polypeptide of SEQ ID NO:
 3. 11.The variant of claim 1, wherein the total number of alterations comparedto SEQ ID NO 3 is 1-20, e.g. 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 alterations.
 12. A method for obtaining a protease variant,comprising introducing into a parent protease a substitution at one ormore positions corresponding to positions 171, 173, 175 or 179 of SEQ IDNO: 3, wherein the variant has an amino acid sequence which is at least70% identical to SEQ ID NO: 3, and recovering the variant. 13-21.(canceled)